WO2008150302A1 - Treatment of neurofibromatosis with radicicol and its derivatives - Google Patents

Treatment of neurofibromatosis with radicicol and its derivatives Download PDF

Info

Publication number
WO2008150302A1
WO2008150302A1 PCT/US2007/075739 US2007075739W WO2008150302A1 WO 2008150302 A1 WO2008150302 A1 WO 2008150302A1 US 2007075739 W US2007075739 W US 2007075739W WO 2008150302 A1 WO2008150302 A1 WO 2008150302A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
hydrogen
deficient
nfl
cells
Prior art date
Application number
PCT/US2007/075739
Other languages
French (fr)
Inventor
Ruihong Chen
Allan E. Rubenstein
Xiaodong Shen
Jin-Chen Yu
Marco Giovannini
Original Assignee
Nexgenix Pharmaceuticals
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2007/070367 external-priority patent/WO2007143630A2/en
Application filed by Nexgenix Pharmaceuticals filed Critical Nexgenix Pharmaceuticals
Priority to CA2727100A priority Critical patent/CA2727100C/en
Publication of WO2008150302A1 publication Critical patent/WO2008150302A1/en
Priority to US12/631,667 priority patent/US8329683B2/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the use of radicicol and its derivatives for treatment of neurofibromatosis type 2 (NF2), neurofibromatosis type 1 (NFl), and neurodegenerative diseases.
  • Neurofibromatosis includes two diseases, neurofibromatosis type 1 (NFl) and neurofibromatosis type 2 (NF2). Both NFl and NF2 are inherited disorders and both encompass mutations which predispose individuals to multiple tumors of the central or peripheral nervous system, and occasionally to other malignancies.
  • Major tumor types associated with NFl and NF2 involve glial cells (e.g. Schwann cells and astrocytes). Although there is the similarity of the involvement of Schwann cells in NFl and NF2 tumors, NFl and NF2 have a spectrum of tumors which involve different types of cells. In addition, NFl and NF2 are caused by different gene mutations.
  • Neurofibromatosis type 2 is a rare form of neurofibromatosis, which is a dominantly inherited tumor suppressor disorder, that affects approximately 1 in 25,000 individuals and is characterized by multiple tumors on the cranial and spinal nerves. NF2 is a different disease from NFl , neurofibromatosis type 1. Although both NFl and NF2 are tumor predisposition syndromes in the nervous system, the tumor suppressor genes are different and signaling pathways are likely to be different. [0005] Individuals with NF2 are at a high risk for developing brain tumors, in particular tumors on both the seventh and eighth cranial nerves. Bilateral vestibular schwannomas, a type of tumor which occurs on these nerves, occurs in about 95% of affected individuals. Consequently, hearing loss, ringing in the ears, and problems with balance are symptoms frequently associated with NF2.
  • Schwannomas are tumors consisting of nerve sheath cells or Schwann cells (SCs). Schwann cells support and protect nerve cells and provide nerves with the insulation they need to conduct info ⁇ nation. Bilateral vestibular schwannomas, also known as acoustic neuromas, as well as spinal schwannomas and schwannomas of the peripheral nerves are common manifestations of NF2. The symptoms of a schwannoma will depend on its location.
  • NF2 may develop other types of tumors emanating from the nerves, meningeal envelopes, brain and spinal cord.
  • the most common tumor of this type is meningioma; other less common tumors include ependymomas and astrocytomas.
  • NF2 patients may have an increased risk for developing mesotheliomas.
  • NF2 is an autosomal dominant genetic trait, meaning it affects both genders equally and each child of an affected parent has a fifty percent chance of inheriting the gene. NF2 results from a mutation or a deletion of the NF2 gene and is transmitted on chromosome 22 (Sainz e/ fl/., 1994, Hum. MoI. Genet. 3: 885-891 ; Ruttledge et al., 1994, Nat. Genet. 6: 180- 184; Rubio et al., 1994, Cancer Res. 54: 45-47; Huynh et al, 1997, J. Neuropathol. Exp. Neurol.
  • the NF2 gene is a tumor suppressor gene that encodes a 595-amino acid protein, termed Merlin.
  • Merlin belongs to the ezrin, radixin, and moesin (ERM) family of proteins (Trofatter et al., 1993, Cell. 75: 826).
  • Neuregulin growth factors (EGF family of growth factors), VEGF, and HGF are important mitogens for Schwann cells (SCs) (Krasnoselsky et al, 1994, J. Neurosci. 14:7284-7290; DeClue et al, 2000, J. Clin. Invest. 105(9): 1233-1241 ; Caye-Thomasen et al, 2005, Otol. Neurotol. 26(l):98-101 ).
  • Neuregulin/ErbB pathways are constitutively activated in human NF2 vestibular schwannomas and inhibitors of these pathways (e.g.
  • Rho-PAK p21 -activated kinase pathway
  • Rac is a member of the Rho family of small GTPases, which organize the actin cytoskeleton and control many cellular processes such as cell proliferation, transformation, and cell motility (Etienne-Manneville and Hall, 2002, Nature. 420(6916): 629-635; Sahai and Marshall, 2002, Nat. Rev. Cancer 2(2): 133-142).
  • PAK can phosphorylate S518 of Merlin (Xiao et al, 2002, J. Biol. Chem. 277: 883-886; Kissel et al, 2002, J. Biol.
  • NFl Neurofibromatosis type 1
  • NFl is one of the most common single gene disorder to affect the human nervous system, with an incidence of 1 in 3500 individuals (Sorensen SA, Mulvihill JJ, Nielsen A. Ann N Y Acad Sci 1986;486:30-7.).
  • NFl affects approximately 1.5 million people worldwide and there is no racial, ethnic, or geographic predilection for the disease.
  • NFl is an autosomal dominantly inherited genetic disorder with frequent gemiline deletion or loss-of- function mutations of the NFl gene, and is caused by mutation in the NFl gene, which encodes Neurofibromin, a tumor suppressor.
  • Neurofibromin shares a region of similarity with the pl20RasGAP protein, therefore functioning as a negative regulator of the Ras pathway.
  • a high spontaneous mutation rate (50%) at the NFl locus and the substantial variability of its expression ensure that the disorder is unlikely to decrease significantly in the population due to genetic screening.
  • the signs of NFl include cafe-au-lait macules, skin freckling, skeletal defects, learning disability, Lisch nodules, dermal and plexiform neurofibromas (most common), benign tumors of the brain or other organs (e.g. optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ganglioneuromas, ependymomas, pheochromocytomas and ganglioneuromas), and malignant neoplasms (e.g.
  • rhabdomyosarcomas neurofibrosarcomas or malignant peripheral nerve sheath tumors
  • MPNST malignant peripheral nerve sheath tumors
  • JMML leukemia-juvenile myelomonocytic leukemia
  • Dermal neurofibromas, subdermal neurofibromas, plexiform neurofibromas and MPNSTs are primarily derived from Schwann cells or their progenitors.
  • Optic gliomas and astrocytomas are derived from astrocytes.
  • Pheochromocytomas are derived from neural crest components (as are neurofibromas and MPNSTs).
  • NFl 53412 v2 DC A
  • the typical characteristic of NFl is the neurofibroma, of which there are clinically and histologically distinct types.
  • Ninety- five% of patients have discrete benign neurofibromas within the dermis which may develop at any time in life, but their numbers are usually small before puberty.
  • the total number of neurofibromas seen in adults varies from just a few to hundreds or even thousands. These tumors may cause disfigurement, chronic pain and pruritus.
  • Certain patients may develop some of the same disfiguring symptoms that are associated with Elephant Man ' s disease, a separate disorder originally thought to be NFl .
  • Plexiform neurofibromas may be congenital and are present in 30% of patients with NFl .
  • NFl neurofibroma
  • plexiform neurofibromas transform to malignant peripheral nerve-sheath tumors, which have a significant mortality rate.
  • NFl is usually not a lethal disorder, affected individuals often face a lifetime of morbidity and disfigurement.
  • the NfI gene was identified in 1990 (Wallace et al. 1990 Science 249: 181-186; Cawthon et al.
  • GTPase-activating protein (GAP) domain of p!20RasGAP (Marchuk et al., 1991 Genomics 1 1 :931-940; Gutmann et al., 1991. Proc. Natl. Acad. Sci. U. S. A. 88: 9658-9662; DeClue et al., 1991. Proc. Natl. Acad. Sci. U. S. A. 88:9914-9918; Martin et al., 1990.
  • GAP GTPase-activating protein
  • Ras-GTP levels from a few NFl MPNST-derived cell lines ST88-14, 88-3 and 90-8 are much higher compared to other cell lines with normal neurofibromin. These cell lines also have activated downstream MAP kinase pathways.
  • cell proliferation and soft agar growth of ST88-14 can be inhibited by injection of an antibody against Ras and expression of the GAP domain of neurofibromin, respectively. Therefore, controlling Ras pathway activity in these cells is important in blocking the transformation properties.
  • the heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone whose function is to ensure the proper folding and stability of a number of its client proteins
  • HSP90 belongs to the ATPase superfamily and consists of three protein domains: the N-terminal ATPase domain, a middle domain responsible for client protein binding, and a C-terminal dimerization domain which also contains a weak ATP-binding domain (Pearl and Prodromou, 2001 Adv Protein Chem. 59: 157-186).
  • Four genes of the HSP90 family are found in humans and their gene products have different cellular locations.
  • HSP90 alpha and HSP90 beta HSP90 alpha and HSP90 beta (Hickey et al., 1989, MoI Cell Biol. 9: 2615-2626).
  • Other major isoforms are GRP94 in the endoplasmic reticulum (Argon and Simen, 1999, Semin, Cell Dev. Biol. 10: 495-505) and TRAP1/HSP75 in mitochondria (Felts et al., 2000, J. Biol. Chem. 275: 3305-12).
  • HSP 90 is found to be part of a series of dynamic multiprotein complexes made of co-chaperones including HSP70, HSP40, and Hop.
  • HSP90 Hydrolysis of ATP causes HSP90 to alter its conformation and allows other co- chaperones such as p23, CDC37, or imunophilins to associate with HSP90 to form a mature complex, which catalyzes the folding and maturation of the client proteins (Pearl & Prodromou, 2000, Curr. Opin. Struct. Biol. 10: 46-51.).
  • the adaptor co-chaperone protein CDC37 mediates interactions between HSP90 and kinases (Pearl, 2005, Curr. Opin. Genet. Dev. 15:55-61 ; Roe et al., 2004, Cell 1 16: 87-98.).
  • HSP90 client proteins There are over one hundred HSP90 client proteins reported in the literature (Solit and Rosen, 2006, Curr. Top. Med. Chem. 6: 1205-14).
  • Major HSP90 client proteins include steroid hormone receptors such as the androgen, estrogen and glucocorticoid receptors (AR, ER, and GR) (Whitesell and Cook, 1996, MoI. Endocrinol. 10: 705-712; Segnitz and Gehring, 1997, J Biol Chem. 272(30): 18694-701 ; Czar et al., 1997 Biochemistry.
  • tyrosine and serine/threonine kinases such as HER2 (ErbB2) (Munster et al., 2002, Cancer Res. 62: 3132-3137.), the insulin-like growth factor- 1 receptor (IGF-IR) (Sepp- Lorenzino et al., 1995, J. Biol. Chem. 270: 16580-16587.), Met (Webb et al., 2000, Cancer Res. 60: 342-349.), Flt-3 (Yao et al., 2003, Clin. Cancer Res.
  • IGF-IR insulin-like growth factor- 1 receptor
  • HSP90 client proteins are important in controlling cell growth and proliferation, differentiation, and cell survival. This is a putative rationale for the use of HSP90 inhibitors in the treatment of cancer. This subject has been extensively reviewed in the recent literature (Chiosis and Neckers, 2006, ACS. Chem. Biol. l(5):279-284; Janin, 2005, J. Med. Chem. 48(241:7503-7512; Sharp and Workman, 2006, Adv. Cancer Res.
  • HSP90 inhibitors More compounds have been found to inhibit the function of HSP90 and are generally referred to as HSP90 inhibitors. Most HSP90 inhibitors inhibit the intrinsic ATPase activity by binding to the N-terminal nucleotide binding site of HSP90 and thus block the formation of the mature complex between HSP90, co-chaperones, and the client proteins since the formation of the mature complex is dependent on ATP hydrolysis.
  • the client proteins are then degraded through the ubiquitin-proteasome degradation pathway (Connell et al., 2001 , Nat. Cell Biol. 3: 93-96.).
  • the coumarin antibiotic novobiocin binds to the C-terminal ATP-binding site of HSP90 but with a very weak activity to degrade HSP90 client proteins (Marcu et al., 2000, J. Natl. Cancer Inst. 92:242-248; Marcu et al., 2000, J. Biol. Chem. 275:37181-37186).
  • analogs of radicicol such as oxime derivatives of radicicol
  • analogs of geldanamycin such as 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG)
  • hydroquinone analogue of 17-AAG have been synthesized and tested.
  • HSP90 inhibitors in various compound classes have been developed as potential agents for cancer treatment. These include purine-based compounds (PCT publications WO/2006/084030; WO/2002/036075; US7,138,401 ; US20050049263; Biamonte et al., 2006, J. Med. Chem. 49:817-828; Chiosis, 2006, Curr. Top. Med. Chem. 6: 1183-1191 ; He et al., 2006, J. Med. Chem.
  • HDAC inhibitors such as Trichostatin A, SAHA, and FK228 are capable of inhibiting deacetylation of HSP90 and thus modulating the function of HSP90 (Kovacs et al., 2005, Molecular Cell, 18: 601-607).
  • Other molecules modulating the level of HSPs such as HSP70 and HSP27 may also affect the function of the HSP90 complex (Zaarur et al., 2006, Cancer Res. 66(3): 1783-1791). None of these HSP90 inhibitors have previously been shown to inhibit the growth of NFl- or NF2- deficient tumor cells.
  • NF2- and NFl -associated tumors consist of surgical removal and focused-beam radiation. Neither treatment is considered optimal. Most patients with NF2 and NFl require multiple surgical and/or focused beam radiation procedures during their lifetime. Since the tumors of NF2 most frequently lie on nerves near the brain and spinal cord, their surgical removal is not without risk. For instance, surgical removal of vestibular schwannomas typically results in complete hearing loss and frequent facial nerve damage. Focused-beam radiation also has a significant incidence of hearing loss and facial nerve damage. Accordingly, a strong need exists for safer treatment options for NF2 and associated tumors (e.g.
  • NFl and associated tumors e.g. dermal neurofibromas, subdermal neurofibromas, plexiform neurofibromas, MPNSTs, gliomas, astrocytomas, and JMML.
  • the present invention provides a novel method for treating NF2 and related NF2-deficient tumors and NFl and related NFl -deficient tumors and their associated signs and symptoms by administering radicicol or its derivatives.
  • the present invention provides a method of treating, preventing or ameliorating tumors or symptoms resulting from neurofibromatosis in a subject suffering neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or neurofibromatosis type 1 (NFl) or a condition associated with the loss of NFl function comprising administering to said subject a therapeutically effective amount of at least one compound of formula I, II, III, IV or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof.
  • NF2 neurofibromatosis type 2
  • NFl neurofibromatosis type 1
  • the present invention provides use of a compound of formula I, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof, for the manufacture of a medicament for the treatment, prevention, or amelioration of tumors or symptoms resulting from neurofibromatosis in a subject suffering neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or neurofibromatosis type 1 (NFl ) or a condition associated with the loss of NFl function.
  • NF2 neurofibromatosis type 2
  • NFl neurofibromatosis type 1
  • the present invention provides a method of treating, preventing or ameliorating a neurodegenerative disease in a patient comprising administering to said patient a therapeutically effective amount of at least one compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof.
  • the present invention provides use of a compound of formula I,
  • the present invention provides a method of inhibiting or reducing the growth or number of NF2-deficient tumor cells or NFl -deficient tumor cells comprising contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with at least one compound of formula I, II, III, IV or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof.
  • R 2 , R J , R 4 and R ""1 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkyl aryl, aralkyl, aryl, hetero alkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH 2 , NR 2 , SR, S(O)R, S(O) 2 R, -S(O) 2 NR 2 , -N(R)SO 2 R, -NC(O)R, -
  • B 1 and B 2 together are -CH 2 -CH 2 - or Bi and B 2 together represent a covalent bond;
  • X 1 is hydrogen, halogen, OH, OR, NH 2 , NR 2 , NH-OR, SR, S(O)R, S(O) 2 R, -N-O- (CH 2 ) H -CO 2 -R; or X 1 together with X 2 or X 3 represents a covalent bond;
  • X 2 and X 3 are both hydrogen, or one of X 2 and X 3 is hydrogen and the other together with X 1 represents a covalent bond;
  • X 6 is hydrogen or X 6 together with one of X 4 and X 3 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl, heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is O, 1 , 2 or 3. [0030]
  • the compound of formula II has the following structure:
  • R is hydrogen, alkyl, arylalkyl, acyl or a protecting group.
  • R' is (CH 2 ) ⁇ C(O)OR, or -(CH 2 ) n C(O)NR 2 ; and n is 0, 1 , 2 or 3.
  • Figure I A shows graphs of Western blots demonstrating NXD30001 downregulates HSP90 client proteins and upregulates HSP70 in NF2-deficient mouse and human cells
  • Figure 1 B shows graphs of Western blots demonstrating that the downregulation of HSP90 client proteins by NXD30001 is time-dependent.
  • Figure 2 shows graphs of actin immunoblots demonstrating the downregulation of HSP90 client proteins in ST88-14 NFl -deficient MPNST cell line resulted by NXD30001.
  • Figure 3 shows the IC50 of radicicol derivatives, namely NXD30001 , NXD30002, and NXD30017, and the IC50 of 17AAG on cell proliferation of NFl and NF2 cells.
  • Tumors in NF2 and NFl patients are unique in that they are slow growing tumors. Both NF2 and NFl tumors have mutations or loss of heterozygosity in tumor suppressor genes although the tumor suppressor genes are different- NF 2 or NFI genes, respectively.
  • the inventors of the present invention have discovered that inhibitors of HSP90 potently blocked the proliferation of NF2-deficient and NFl -deficient tumor cells and also delay the growth of NF2-deficient and NFl -deficient tumors in mice. Merlin regulates the abundance
  • HSP90 HSP90-binding protein kinases
  • AKT receptor tyrosine kinases
  • Raf receptor tyrosine kinases
  • aberrant activation of the PI3K/AKT pathway has been found in human schwannomas from NF2 patients (as compared to normal nerves), in human NF2-def ⁇ cient tumor xenografts (e.g.
  • Neurofibromin is a negative regulator of Ras.
  • Raf the direct downstream effector of Ras, is a well-known client protein of HSP90.
  • Neurofibromin has also been shown to regulate AKT.
  • a compound that inhibits HSP90 will likely be able to reduce the amount of AKT and other HSP90 client proteins such as ErbB2, IGF-IR, and Raf in the NF2-deficient cells or NFl -deficient cells. Reduction of the amount or activity of these proteins may be useful to reduce or stabilize the proliferation of NF2-deficient cells or NFl -deficient cells or to cause apoptosis of NF2-deficient cells or NFl -deficient cells.
  • a compound that inhibits the activity of HSP90 is a compound that directly binds to the HSP90 protein or modifies the HSP90 protein post-translationally or regulate the transcription of HSP proteins such as HSP70 and HSP27 (Zaarur et al., 2006, Cancer Res. 66(3): 1783-1791).
  • HSP90 can be acetylated and inactivated by histone deacetylase (HDAC) inhibitors (Kovacs et al., 2005, MoI. Cell 18(5):601-607; Fuino et al., 2003, MoI. Cancer Ther. 2:971-984; Aoyagi and Archer, 2005, Trends Cell. Biol. 15(1 1):565-567).
  • HDAC histone deacetylase
  • HSP90 inhibitory compounds are determined to be useful for the treatment of NF2-deficient tumors and NFl -deficient tumors which may not respond well to traditional chemotherapy and other cancer therapies which target fast growing and heterogeneous cancer cells.
  • NF2- Associated Tumors NF2- Associated Tumors
  • NF2 neurofibromatosis type-2
  • NF2 neurofibromatosis type-2
  • This genetic characteristic i.e., inactivation of the NF2 gene, differentiates tumors found in NF2 patients from genetically heterogeneous tumors such as breast and colon cancer tumors.
  • NF2 patients have NF2-deficient meningiomas whereas some non-NF2-deficient meningiomas may contain mutations in many different oncogenes or tumor suppressor genes.
  • NF2-deficient tumors refer to tumors which contain a nonfunctioning NF2 gene.
  • a non-functioning NF2 gene can be the result of a one or more insertion or deletion mutations within the NF2 gene, for instance, missense or nonsense mutations, mutations in the promoter or enhancer or introns that lead to no/low expression of
  • NF2-deficient tumors are found in mesotheliomas and in patients with NF2, and include schwannomas, meningiomas, and other tumors associated with the nervous system. NF2-deficient tumors are also found in all patients with sporadic schwannomas and in 50%-70% of patients with meningiomas. [0040] The presence of NF2-deficient bilateral vestibular schwannomas, i.e., Schwann cell tumors, is a hallmark of NF2.
  • the methods of the present invention can be used to inhibit the growth and/or kill NF2-deficient schwannoma cells, including those associated with vestibular schwannomas, spinal cord and other peripheral nerve schwannomas and sporadic schwannomas.
  • NF2-deficient schwannoma cells including those associated with vestibular schwannomas, spinal cord and other peripheral nerve schwannomas and sporadic schwannomas.
  • NF2-deficient tumors refer to tumors which contain a nonfunctioning NF2 gene.
  • Merlin interacts with or regulates, but is not limited to, proteins and pathways such as Paxillin/Integrin- ⁇ l/ErbB2, EGFR, Patched/Smoothened, HRS, CD44, E- Cadherin, Fat, EBP50/NHE-RF/PDGFR, Wingless, Notch, Rac-PAK, PBK-AKT, Ras-Raf- Mek-Erk2, Hippo pathways, and downstream proteins thereof.
  • Figure 1 is a schematic of involvement of Merlin with multiple cell surface proteins and signaling pathways. Targeting multiple proteins or pathways may be necessary for treating NF2.
  • NFl- Associated Tumors such as Paxillin/Integrin- ⁇ l/ErbB2, EGFR, Patched/Smoothened, HRS, CD44, E- Cadherin, Fat, EBP50/NHE-RF/PDGFR, Wingless, Notch, Rac-
  • NFl neurofibromatosis type- 1
  • This genetic characteristic i.e., inactivation of the NFl gene, differentiates tumors found in NFl patients from genetically heterogeneous tumors such as breast and colon cancer tumors. For instance, tumors found in NFl patients all have NFl gene mutations whereas patients with other cancers have mutations in different genes or overexpression of different genes.
  • NFl -deficient tumors refer to tumors which contain a non- functioning NFl gene.
  • a non-functioning NFl gene can be the result of one or more insertion or deletion mutations within the NFl gene, for instance, missense or nonsense mutations, mutations in the promoter or enhancer or introns that lead to no/low expression of the NFl gene, or the deletion of the entire NFl gene.
  • NFl -deficient tumors are found in patients with NFl , and include dermal, subdermal, plexiform neurofibromas, and MPNST and other tumors associated with the nervous system. NFl -deficiency also predisposes individuals to a rare form of leukemia, JMML.
  • NFl diagnosis is confirmed by fulfilling NIH clinical criteria or by finding an NFl mutation with mutational analysis.
  • the methods of the present invention can be used to
  • 53412 v2/DC 14 inhibit the growth and/or kill NFl -deficient tumors, including dermal, subdermal, plexiform neurofibromas, MPNST, gliomas, astrocytomas, pheochromocytomas and JMML.
  • the present invention provides the use of radicicol or its derivatives based on the resorcylic acid lactones, preferably compounds of formula I, II, III, IV or V, as HSP90 inhibitors for the treatment, prevention, or amelioration of tumors or symptoms resulted from neurofibromatosis. Also provided are compositions comprising the compounds and processes for the preparation of the compounds.
  • the compound of fo ⁇ nula I has the following structure:
  • R 2 , R “ ', R 4 and R 5 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkyl aryl, aralkyl, aryl, heteroalkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH 2 , NR 2 , SR, S(O)R, S(O) 2 R, -S(O) 2 NR 2 , -N(R)SO 2 R, -NC(O)R, - NC(O)NR 2 , -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR 2 , -OC(O)OR, or - OC(O)NR 2 ; or R 1 , R 2 , R 3 , and R 5 are independently hydrogen, halogen, nitro,
  • L 1 and L 2 are each independently a covalent bond, -0-, or -NR 3a -;
  • R l a and R 2a are each independently H, alkyl, cyclylalkyl, heteroaryl, heterocyclyl, alkenyl, alkynyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, -alkylene-C(O)-O-R 4a , - (alkylene)-C(O)-O-R 4a , -alkylene-O-C(O)-O-R 4a , or -(alkylene)-O-C(O)-O-R 4a ; and
  • R 3a and R 4a are each independently H, alkyl, cyclylalkyl, heterocyclyl, heteroaryl, alkenyl, alkynyl, arylalkyl, or heterocyclylalkyl;
  • B 1 and B 2 together are -CH 2 -CH 2 - or Bi and B 2 together represent a covalent bond;
  • X 1 is hydrogen, halogen, OH, OR, NH 2 , NR 2 , NH-OR, SR, S(O)R, S(O) 2 R, -N-O- (CH 2 ) H -CO 2 -R; or X 1 together with X 2 or X 3 represents a covalent bond;
  • X 2 and X 3 are both hydrogen, or one of X 2 and X 3 is hydrogen and the other together with X 1 represents a covalent bond;
  • X 6 is hydrogen or X 6 together with one of X 4 and X 3 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl, heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is O, 1 , 2 or 3.
  • R 4 has the structure of formula Ia, which may increase the water solubility and/or bioavailability of the compound of formula I.
  • the compound of formula I has the structure of formula II:
  • R 1 is H, halogen or heterocyclyl.
  • R 5 is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or arylalkyl.
  • a 1 and A 2 together are 1 ,2- oxirane.
  • R 1 is H, Cl or heterocyclyl
  • R 2 and R 4 are independently OH or OR;
  • R 5 is hydrogen, alkyl, aryl or aralkyl;
  • X 1 is hydrogen, halogen or NH-OR; and
  • R 1 is H, Cl or heterocyclyl
  • R 2 and R 4 are independently OH or OR;
  • R 5 is hydrogen, alkyl, aryl or aralkyl;
  • a 1 and A 2 together are 1 ,2-oxirane;
  • X 1 is hydrogen, halogen or NH-OR; and
  • R 1 is H, Cl or heterocyclyl
  • R 2 and R 4 are independently OH or OR;
  • R 5 is hydrogen, alkyl, aryl or aralkyl;
  • X 1 together with X 2 represent a bond; and
  • R 1 is H or Cl
  • R 5 is hydrogen, methyl, propyl, isopropyl or phenyl
  • R 1 is H, Cl or heterocyclyl
  • R 2 and R 4 are independently OH or OR;
  • R 5 is hydrogen, alkyl, aryl or aralkyl;
  • a 1 and A 2 together are 1 ,2-oxirane;
  • X 1 together with X 2 represent a bond; and
  • the compound of formula I has the structure of formula III:
  • R 8 is hydrogen, alkyl, arylalkyl, acyl or a protecting group.
  • R is hydrogen or acyl
  • R ' is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or arylalkyl.
  • X 1 together with X 2 represent a covalent bond.
  • Ai and Ai together are 1 ,2- oxirane.
  • the compound of formula I has the structure of formula IV;
  • R 6 is hydrogen, OR, or NR 2 .
  • R is hydrogen or acyl
  • R 1 is H, halogen or heterocyclyl.
  • R 5 is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or aryl alkyl.
  • a 1 and A 2 together are 1,2- oxirane.
  • the compound of formula I has the structure of formula V:
  • R 9 is (CH 2 ) n C(O)OR, or -(CH 2 ) n C(O)NR 2 ; and n is 0, 1 , 2 or 3.
  • R 6 is -CH 2 C(O)N(Me)OMe.
  • R 1 is H, halogen or heterocyclyl.
  • R 5 is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or arylalkyl.
  • a 1 and A 2 together are 1 ,2- oxirane.
  • -NR 2 can be -N(R X ) 2 , -N(R y ) 2 , or -NR x R y .
  • Nonlimiting examples of pharmaceutically acceptable salts are (a) acid addition salts formed with inorganic acids such as sulfate, nitrate, bicarbonate, and carbonate salts (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids including tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and ⁇ - glycerophosphate salts, such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalcturonic acid; (b) base addition salts formed with inorgan
  • quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula -NR ⁇ A " , wherein R is as defined above and A is a counterion, including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).
  • R is as defined above and A is a counterion, including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succ
  • compositions may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • pharmaceutically acceptable "prodrugs” refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, animated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound.
  • the compounds are prepared in optically active form by asymmetric synthesis using the processes described herein or synthetic transformations known to those skilled in the art.
  • optically active materials examples include at least the following.
  • first- and second-order asymmetric transformations a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer.
  • the desired enantiomer is then released from the diastereomer;
  • kinetic resolutions this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions;
  • enantiospecific synthesis from non-racemic precursors a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis;
  • chiral liquid chromatography a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase.
  • the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions;
  • chiral gas chromatography a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;
  • xii) extraction with chiral solvents a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; or
  • xiii) transport across chiral membranes a technique whereby a racemate is placed in contact with a thin membrane barrier.
  • the barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through.
  • the compound means one or more compounds of formula I, II, III, IV, or V, or a pharmaceutically acceptable tautomer, salt, solvate, prodrug, or physiologically functional derivative thereof.
  • a compound of formula (number) means a compound of that formula and
  • Ester thereof means any ester of a nicotinic desensitizer in which any of the - COOH functions of the molecule is replaced by a -COOR function, in which the R moiety of the ester is any carbon-containing group which forms a stable ester moiety, including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and substituted derivatives thereof.
  • esters thereof includes but is not limited to pharmaceutically acceptable esters thereof
  • Salt thereof means any acid and/or base addition salt of a compound of the present invention; preferably a pharmaceutically acceptable salt thereof.
  • “Pharmaceutically acceptable salt” means a salt of a compound of the present invention which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, generally water or oil-soluble or dispersible, and effective for their intended use. Where applicable and compatible with the chemical properties of the nicotinic desensitizer, the term includes pharmaceutically-acceptable acid addition salts and pharmaceutically-acceptable base addition salts.
  • solute a nicotinic desensitizer formed by solvation (the combination of solvent molecules with molecules or ions of the solute), or an aggregate that consists of a solute ion or molecule (the nicotinic desensitizer) with one or more solvent molecules.
  • Chiral refers to molecules which have the property of non-superimposability of the mirror image partner, while the term '"achiral " refers to molecules which are superimposable on their mirror image partner.
  • Tautomers refer to isomers of nicotinic desensitizers in which the isomers change into one another with great ease so that they ordinarily exist together in equilibrium.
  • Stepoisomers refer to nicotinic desensitizers which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • Diastereomer refer to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
  • Enantiomers refer to two stereoisomers of a nicotinic desensitizer which are non- superimposable mirror images of one another.
  • d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
  • a compound prefixed with (+) or d is dextrorotatory.
  • these stereoisomers are identical except that they are mirror images of one another.
  • a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
  • the terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
  • protecting groups include prodrug moieties and chemical protecting groups.
  • Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e. routes or methods to prepare the compounds of the invention. For the most part the decision as to which groups to protect, when to do so, and the nature of the chemical protecting group "PG" will be dependent upon the chemistry of the reaction to be protected against (e.g., acidic, basic, oxidative, reductive or other conditions) and the intended direction of the synthesis. The PG groups do not need to be, and generally are not, the same if the compound is substituted with multiple PG.
  • PG will be used to protect functional groups such as carboxyl, hydroxyl, thio, or amino groups and to thus prevent side reactions or to otherwise facilitate the synthetic efficiency.
  • the order of deprotection to yield free, deprotected groups is dependent upon the intended direction of the synthesis and the reaction conditions to be encountered, and may occur in any order as determined by the artisan.
  • Various functional groups of the compounds of the invention may be protected. For example, protecting groups for -OH groups (whether hydroxyl, carboxylic acid, phosphonic
  • Ether- or ester-forming groups are capable of functioning as chemical protecting groups in the synthetic schemes set forth herein. However, some hydroxyl and thio protecting groups are neither ether- nor ester- forming groups, as will be understood by those skilled in the art, and are included with amides, discussed below.
  • Ester-forming groups include: (1) phosphonate ester-forming groups, such as phosphonamidate esters, phosphorothioate esters, phosphonate esters, and phosphon-bis- amidates; (2) carboxyl ester-forming groups, and (3) sulphur ester-forming groups, such as sulphonate, sulfate, and sulfinate.
  • phosphonate ester-forming groups such as phosphonamidate esters, phosphorothioate esters, phosphonate esters, and phosphon-bis- amidates
  • carboxyl ester-forming groups such as sulphonate, sulfate, and sulfinate.
  • the invention includes compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof.
  • Such products typically are identified by preparing a radiolabeled (e.g., C 14 or H J ) compound of the invention, administering it parenterally in a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples.
  • a detectable dose e.g., greater than about 0.5 mg/kg
  • an animal such as rat, mouse, guinea pig, monkey, or to man
  • sufficient time for metabolism to occur typically about 30 seconds to 30 hours
  • isolating its conversion products from the urine, blood or other biological samples typically isolating its conversion products from the urine, blood or other biological samples.
  • a term in the specification is identified as a range (i.e. Ci -4 alkyl), the range independently refers to each element of the range.
  • C] -4 alkyl means, independently, Ci, C 2 , C 3 or C 4 alkyl.
  • substituents when one or more substituents are referred to as being "'independently selected from' " a group, this means that each substituent can be any element of that group, and any combination of these groups can be separated from the group.
  • R 1 and R 2 can be independently selected from X, Y and Z, this separately includes the groups R 1 is X and R 2 is X; R 1 is X and R 2 is Y; R 1 is X and R 2 is Z; R 1 is Y and R 2 is X; R 1 is Y and R 2 is Y; R 1 is Y and R 2 is Z; R 1 is Z and R 2 is X; R 1 is Z and R 2 is Y; and R 1 is Z and R 2 is Z.
  • alkyl refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, including but not limited to groups with C 1 to C) 0 .
  • lower alkyl refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, including groups with Ci to C 4 , and if appropriate a cyclic alkyl group (for example cyclopropyl).
  • alkyl groups are methyl, ethyl, propyl, wopropyl, cyclopropyl, butyl, secbutyl, isobutyl, tertbutyl, cyclobutyl, 1-methylbutyl, 1 ,1- dimethylpropyl, pentyl, cyclopentyl, isopentyl, neopentyl, cyclopentyl, hexyl, isohexyl, and cyclohexyl.
  • the alkyl group can be unsubstituted or substituted with one or more moieties selected from the group consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, thiol, imine, sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfmyl, sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acid halide, anhydride, oxime, hydrazine, carbamate, phosphonic acid, phosphate, phosphonate, or any other viable functional group that does not inhibit
  • 'halo or "halogen”, as used herein, includes chloro, bromo, iodo, and fluoro.
  • alkyl thio refers to a straight or branched chain alkylsulfide of the number of carbons specified, such as for example, C 1 . 4 alkyltb.io, ethylthio, -S-alkyl, -S-alkenyl, -S- alkynyl, etc.
  • alkylamino'Or “arylamino” refer to an amino group that has one or two alkyl or aryl substituents, respectively. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, then it is a lower alkyl, whether substituted or unsubstituted.
  • alkylsulfonyl means a straight or branched alkylsulfone of the number of carbon atoms specified, as for example, Ci -6 alkylsulfonyl or methylsulfonyl.
  • alkoxycarbonyl refers to a straight or branched chain ester of a carboxylic acid derivative of the number of carbon atoms specified, such as for example, a methoxycarbonyl, MeOC(O)-.
  • nitro means -NO 2
  • 'sulfhydryl means -SH
  • sulfonyl means -S(O)T.
  • alkenyl and alkynyl refer to alkyl moieties, including both substituted and unsubstituted forms wherein at least one saturated C-C bond is replaced by a double or triple bond.
  • C 2-6 alkenyl may be vinyl, allyl, 1-propenyl, 2-propenyl, 1 -butenyl, 2- butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, or 5-hcxenyl.
  • C 2-6 alkynyl may be ethynyl, 1 -propynyl, 2- propynyl, 1 -butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1 - hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl.
  • alkyl ene includes a saturated, straight chain, divalent alkyl radical of the formula -(CH 2 ) H -, wherein "n " may be any whole integer from 1 to 10.
  • alkyl includes both straight chain and branched groups. However, reference to an individual radical such as “propyl” embraces only that straight-chain radical, whereas a branched chain isomer such as “isopropyl” is specifically termed such.
  • aryl refers to any stable monocyclic, bicyclic, or tricyclic carbon ring of up to 8 members in each ring, wherein at least one ring is aromatic as defined by the Huckel 4n+2 rule, and especially phenyl, biphenyl, or naphthyl. The term includes both substituted and unsubstituted moieties.
  • the aryl group can be substituted with any described moiety, including but not limited to one or more moieties
  • 53412 v2/DC 52 selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, azido, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either protected or unprotected as necessary, as known to those skilled in the art, for example, as taught in Greene et al., Protective Groups in Organic Synthesis. John Wiley & Sons, 3 ld Ed., 1999.
  • alkaryl or "'alkylaryl” refers to an alkyl group with an aryl substituent or an alkyl group linked to the molecule through an aryl group as defined herein.
  • aralkyl or “arylalkyl” refers to an aryl group substituted with an alkyl substituent or linked to the molecule through an alkyl group as defined above.
  • cycloalkyl includes a ring Of C 3-8 , including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • alkoxy means a straight or branched chain alkyl group having an attached oxygen radical, the alkyl group having the number of carbons specified or any number within this range.
  • alkyl group having the number of carbons specified or any number within this range.
  • a "-O-alkyl' " C 1 .4 alkoxy, methoxy, etc.
  • acyl " ' or "'O-linked ester” includes a group of the formula C(O)R " , wherein R' is an straight, branched, or cyclic alkyl (including lower alkyl), carboxylate residue of an amino acid, aryl including phenyl, heteroaryl, alkaryl, aralkyl including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl (including lower alkyl), aryl including phenyl optionally substituted with chloro, bromo, fluoro, iodo, Ci to C 4 alkyl or Q to C 4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxy-trityl, substituted benzyl, al
  • Aryl groups in the esters optimally comprise a phenyl group.
  • acyl groups include acetyl, trifluoroacetyl, methylacetyl, cyclopropyl acetyl, cyclopropyl- carboxy, propionyl, butyryl, isobutyryl, hexanoyl, heptanoyloctanoyl, neo-heptanoyl, phenylacetyl, 2-acetoxy-2- phenylacetyl, diphenylacetyl, ⁇ -methoxy- ⁇ -trifluoromethyl-phenylacetyl, bromoacetyl, 2- nitro-benzeneacetyl, 4-chloro-benzeneacetyl, 2-chloro-2,2-diphenylacetyl, 2-chloro-2- phenylacetyl, trimethylacetyl, chlorodifluoroacetyl, per
  • carbonyl includes a group of the structure "-C(O)-X-R " or "X-C(O)-R “ ', where X is O, S, or a bond, and each R is independently as defined above.
  • heteroatom includes an atom other than carbon or hydrogen in the structure of a heterocyclic compound, nonlimiting examples of which are nitrogen, oxygen, sulfur, phosphorus or boron.
  • heterocycle includes non-aromatic ring systems having four to fourteen members, preferably five to ten, in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom.
  • heterocyclic rings include 3-l H-benzimidazol-2-one, (l -substituted)-2-oxo- benzimidazol-3-yl, 2-tetrahydro-furanyl, 3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3- tetrahydropyranyl, 4-tetra-hydropyranyl, [l ,3]-dioxalanyl, [l ,3]-dithiolanyl, [l ,3]-dioxanyl, 2- tetra-hydro-thiophenyl, 3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl, 4- morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2- pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1
  • heterocyclyl or “heterocyclic”, as it is used herein, is a group in which a non-aromatic heteroatom- containing ring is fused to one or more aromatic or non-aromatic rings, such as in an indolinyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the non-aromatic heteroatom-containing ring.
  • heterocycle or “heterocyclic” whether saturated or partially unsaturated, also refers to rings that are optionally substituted.
  • heteroaryl used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroaryl alkoxy”, refers to heteroaromatic ring groups having five to fourteen members.
  • heteroaryl rings examples include 2-furanyl, 3-furanyl, 3-furazanyl, N-imidazolyl, 2- imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3- pyi ⁇ olyl, 1-pyrazolyl, 2-pyi'azolyl, 3-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-pyriniidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2- triazolyl, 5-triazolyl, 2-thienyl
  • heteroaryl is a group in which a heteroatomic ring is fused to one or more aromatic or nonaromatic rings where the radical or point of attachment is on the heteroaromatic ring. Examples include tetrahydroquinolinyl, tetrahydroisoquino-linyl, and pyrido [3,4-d]pyrimidinyl.
  • heteroaryl also refers to rings that are optionally substituted.
  • heteroaryl may be used interchangeably with the term “heteroaryl ring” or the term “heteroaromatic”.
  • amino as used herein unless otherwise specified, includes a moiety represented by the structure "-NR 2 ", and includes primary, secondary and tertiary amines optionally substituted by alkyl, aryl, heterocyclyl, and/or sulfonyl groups.
  • R 2 may represent two hydrogen atoms, two alkyl moieties, or one hydrogen and one alkyl moiety.
  • amido as used herein includes an amino-substituted carbonyl, while the term “amidino” means a group having the structure "-C(NH)-NH 2 ".
  • quaternary amine as used herein includes quaternary ammonium salts that have a positively charged nitrogen. They are formed by the reaction between a basic nitrogen in the compound of interest and an appropriate quaternizing agent such as, for example, methyliodide or benzyliodide. Appropriate counterions accompanying a quaternary amine include acetate, trifluoroacetate, chloro, bromo and iodo ions.
  • substituted includes multiple degrees of substitution by one or more named substituents such as, for example, halo, hydroxyl, thio, alkyl, alkenyl, alkynyl, nitro, cyano, azido, amino, carboxamido, etc.
  • substituents such as, for example, halo, hydroxyl, thio, alkyl, alkenyl, alkynyl, nitro, cyano, azido, amino, carboxamido, etc.
  • substituents such as, for example, halo, hydroxyl, thio, alkyl, alkenyl, alkynyl, nitro, cyano, azido, amino, carboxamido, etc.
  • biological sample includes, without limitation, cell cultures or extracts thereof; preparations of an enzyme suitable for in vitro assay; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.
  • pharmaceutically acceptable carrier refers to a non-toxic earner, adjuvant, or vehicle that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.
  • the double bond geometry can be either E or Z. Both the E and Z forms of the double bond geometry fall within the scope of the present invention.
  • the present invention includes methods of inhibiting the growth of NF2-deficient and/or NFl -deficient tumor cells by contacting, i.e., treating, the NF2-deficient or NFl - deficient tumor cells with radicicol and its derivatives, such as one or more compounds of formula I, II, III, IV, or V as described above.
  • the present invention also includes methods of decreasing proliferation of NF2-deficient or NFl -deficient tumor cells by contacting the cells with radicicol and its derivatives, such as one or more compounds of formula I, II, III, IV, or V as described above.
  • inhibition of the growth of NF2 or NFl cells is determined by comparing a sample of NF2-deficient or NFl -deficient tumor cells treated with radicicol and its derivatives to a control, such as a sample of untreated NF2-deficient or NFl -deficient tumor cells or a sample of cells treated with a known inert compound.
  • a control such as a sample of untreated NF2-deficient or NFl -deficient tumor cells or a sample of cells treated with a known inert compound.
  • a control such as a sample of untreated NF2-deficient or NFl -deficient tumor cells or a sample of cells treated with a known inert compound.
  • a control Prior to contacting cells with radicicol and its derivatives, both samples of NF2-deficient cells or NFl-deficient cells (treated and control) should consist of approximately the same number of cells and be of the same cell type, e.g., NF2-deficient schwannomas or
  • 53412 v2 ⁇ DC 55 may decrease in number by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% following compound treatment compared to the control.
  • Radicicol a macrocyclic lactone antibiotic
  • Radicicol analogs ' or "radicicol derivatives" as used herein denotes macrocyclic lactone compounds that are structurally similar to radicicol.
  • the "radicicol analogs' " or “radicicol derivatives " ' refer to compounds of fused bicyclic ring structure wherein a six-membered aromatic ring shares two carbon atoms with a 12- to 16-membered non-aromatic ring containing a lactone group and at least one olefin group in the core of the 12- to 16-membered ring.
  • the radicicol analogs/derivatives may have one or more substituents on the six- membered aromatic ring or the 12- to 16-membered non-aromatic ring. It is noted that the terms “analog” and “derivative” are used interchangeably in the present application.
  • radicicol and derivatives thereof have a structure of formula I, II, III, IV, or V as described above.
  • radicicol and derivatives thereof are used in combination with at least one additinoal active agent to inhibit the growth of NF2- deficient and/or NFl -deficient tumor cells.
  • the at least one additional active agent is one or more HSP90 inhibitors. That is, the methods of inhibiting the growth of NF2- deficient and/or NFl -deficient tumor cells further comprises contacting, i.e., treating, the NF2-deficient or NFl -deficient tumor cells with at least one additinoal active agent, such as one or more HSP90 inhibitors.
  • the compound of the present invention contacts the NF2-deficient or NF 1 -deficient tumor cells with another active agent.
  • HSP90 functions as a chaperone for its client proteins as part of dynamic multiprotein complexes, here define as " ⁇ SP90 complexes " or "'HSP90 chaperone machineries, which consist of co-chaperones such as HSP90, HSP70, HSP27, HSP40, HOP, p23, and CDC37 and individual client protein.
  • ⁇ SP90 complexes or "'HSP90 chaperone machineries, which consist of co-chaperones such as HSP90, HSP70, HSP27, HSP40, HOP, p23, and CDC37 and individual client protein.
  • a HSP90 inhibitor refers to a substance that inhibits or reduces the growth of or number of NF2-deficient tumor cells or NFl -deficient tumor cells by inhibiting HSP90 function in these cells.
  • Any compound that modulates the activity of HSP90 i.e., a HSP90 modulator, may also be used to inhibit the growth of NF2- deficient tumor cells.
  • a HSP90 modulator may modulate HSP90 by inhibiting or down-regulating, i.e., reducing, the activity of one or more proteins of the HSP90 complex.
  • biological agents or molecules that inhibit or reduce the activity of the HSP90 complex may also be used to inhibit the growth of NF2-deficient or NFl -deficient tumor cells.
  • biological agents or molecules of the present invention include, but are not limited to antibodies, peptides, siRNAs, antisense nucleic acids, and any combinations thereof.
  • Inhibition by a HSP90 inhibitor can occur through a reduction in the capability of the HSP90 protein or complex to assist its client proteins to function in or to activate a signaling
  • the HSP90 inhibitor may reduce the amount of total or phosphorylated forms of specific client proteins of HSP90 which in turn inhibits the activity of the downstream pathways of these client proteins.
  • the HSP90 inhibitor/modulator can upregulate the amount of the HSP70 protein and other HSP proteins in the HSP90 complex.
  • HSP90 function can be inhibited or modulated, for example, such as by binding of a compound as disclosed herein to HSP90 or by modifying the HSP90 protein postranslationally. Also, such as by inhibition of a kinase activity of a protein, by inhibition of dimerization of a protein, inhibition of DNA binding of a protein, or inhibition of transactivation of a protein.
  • HSP90 inhibitors can inhibit one or more proteins in the HSP90 complex by an antibody specific for it or by employing antisense, siRNA, or ribozyme technologies to reduce the level of mRNA coding for the protein.
  • the HSP90 inhibitors or modulators useful for the present invention include, but are not limited to benzoquinone-based ansamycin antibiotics, purine scaffold-based HSP90 inhibitors, pyrazole or imidazole scaffold-based HSP90 inhibitors, tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitors, novobiocin and its analogs, and any combinations thereof.
  • HSP90 inhibitors are benzoquinone-based ansamycin antibiotics, the structure of which typically contain a 1 ,4-benzoquinone ring fused with a 18- to 20-membered non-aromatic ring containing a lactam group and at least one olefin group in the core of the 18- to 20-membered ring.
  • WO 98/51702 which is herein incorporated by reference in its entirety
  • Some representative benzoquinone-based ansamycin antibiotics are shown in Scheme 2A. [00164] SCHEME 2A
  • purine scaffold-based compounds are HSP90 inhibitors.
  • HSP90 inhibitors See, for example, WO 02/36705, WO 03/037860, and WO 2006/084030, all of which are herein incorporated by reference in their entirety
  • These purine scaffold-based HSP90 inhibitors typically have a structure wherein an adenine ring and a six-membered aryl or heteroaryl ring are linked through a linker which can be methylene, fluorinated methylene, sulfur, oxygen, nitrogen, carbonyl, imine, sulfmyl, or sulfonyl.
  • Scheme 3A shows two compounds exemplifying the purine scaffold-based HSP90 inhibitors.
  • SCHEME 3A shows two compounds exemplifying the purine scaffold-based HSP90 inhibitors.
  • pyrazole or imidazole scaffold-based compounds are known to inhibit HSP90. These pyrazole or imidazole scaffold-based HSP90 inhibitors are typically non-fused tricyclic compounds wherein two aryl or heteroaryl rings are attached to two adjacent positions (carbon or nitrogen atom) of a pyrazole or imidazole ring, respectively. (See, for example, WO 2007/021877, which is herein incorporated by reference in its entirety, or Vernalis Ltd, Bioorg Med Chem Lett, 2006, 16, 2543-2548, or Sharp et al., Molecular Cancer Therapeutics, 2007, 6, 1 198-121 1). Examples of pyrazole or imidazole scaffold-based HSP90 inhibitors are shown in Scheme 4A. [00168] SCHEME 4A
  • HSP90 inhibitors are tetrahydroindolone and tetrahydroindazolone derivatives reported in WO 2006/091963, the disclosure of which is herein incorporated by reference in its entirety. These tetrahydroindolone or tetrahydroindazolone based HSP90 inhibitors generally have a structure wherein a substituted aryl group is directly attached to the nitrogen atom of a tetrahydroindolone or tetrahydroindazolone.
  • Scheme 5 A shows certain examples in WO 2006/091963. [00170] SCHEME 5A
  • HSP90 Many client proteins of HSP90 are kinases which are involved in phosphorylation cascades. For this reason, presence and/or activity of a client protein of HSP90 can be assessed by measuring the phosphorylation of the protein and/or a downstream protein. Inhibition of phosphorylation or hypophosphorylation is an indication of a decrease or inhibition of activity of a protein.
  • the proliferation of NF2-deficient tumor cells or NFl -deficient cells can be reduced or stabilized by the administration of a HSP90 inhibitory compound that inhibits or reduces the phosphorylation of one or more proteins of the PI3K/Akt pathway or Ras-Raf pathway including, but not limited to, Akt, mTOR, FKHR, GSK3, S6, S6K, Bad, Raf, Mek, and Erkl/2 proteins.
  • the proliferation of NF2-deficient or NFl- deficient tumor cells can be reduced or stabilized by the administration of a compound of the present invention and/or a HSP90 inhibitory compound that reduces or degrades ErbB2 or phospho-ErbB2.
  • the proliferation of NF2-deficient or NFl - deficient tumor cells can be reduced or stabilized by the administration of a compound of the present invention and/or a HSP90 inhibitory compound that reduces or degrades other receptor tyrosine kinases.
  • a cytotoxic agent and a compound of the present invention can also be contacted together with NF2-deficient or NFl -deficient tumor cells, either simultaneously or sequentially.
  • the cytotoxic agent i.e., chemotherapeutic drug
  • Cytotoxic agents that can be co-administered with the compound of the present invention are chemotherapy drugs known in the art.
  • Growth of NF2-deficient or NFl -deficient tumor cells can be inhibited in vitro or ex vivo.
  • NF2-deficient or NFl -deficient tumor cells can be primary cell culture cells or a tissue culture cell line cells.
  • Cells can be mammalian cells, including, but not limited to, human, canine, rat and murine yV/2-deficient or JV/7 -deficient tumor cells.
  • methods of the invention include the use of ⁇ f/2-deficient mouse cells ⁇ e.g., Schwann cells) or NF2- deficient human tumor cells ⁇ e.g., Schwann cells) or NfI -deficient mouse cells ⁇ e.g. Schwann cells and MPNST cells) or NFl -deficient human tumor cells (e.g. MPNST cells, astrocytoma cell, JMML cells).
  • Cells can be confluent or subconfluent when contacted with radicico or its derivatives.
  • NF2-deficient or NFl -deficient tumor cells are contacted with radicicol or its derivatives in vivo.
  • radicicol or its derivatives is
  • NF2-deficient or NFl -deficient tumor cells can be contacted in vitro or ex vivo with very small amounts of compound of the present invention to trigger inhibition of cell growth and/or resulting in cell death.
  • NF2-deficient or NFl -deficient cells can be contacted with at least about .001 ⁇ M of compound of the present invention, at least about .01 ⁇ M of compound of the present invention, at least about .05 ⁇ M of compound of the present invention, at least about 0.08 ⁇ M of compound of the present invention, at least about 0.09 ⁇ M of compound of the present invention, at least about 0.1 ⁇ M of compound of the present invention, at least about 0.2 ⁇ M of compound of the present invention, at least about 0.3 ⁇ M of compound of the present invention, at least about 0.4 ⁇ M of compound of the present invention, at least about 0.5 ⁇ M of compound of the present invention, at least about 0.6 ⁇ M of compound of the present invention, at least about 0.7 ⁇ M of compound of the present invention, at least about 0.8 ⁇ M of compound of the present invention, at least about 0.9 ⁇ M of compound of the present invention, at least about 1 ⁇ M of compound of the present invention, at least about 1.5 ⁇
  • Appropriate amounts of the compound to contact with the cells can be determined by persons skilled in the art to obtain the desired level of inhibition of cell growth or the corresponding amount of cell death.
  • Inhibition of cell growth or cell death can be measured by methods known in the art. For instance, in vitro and ex vivo inhibition of cell growth and cell death can be measured by a cell proliferation assay and a cell apoptosis assay. Cell death or inhibition of proliferation can be measured in terms of the IC50 of the compound of the present invention.
  • the IC50 of NF2-deficient tumor cells treated in vitro or ex vivo is at least about .001 ⁇ M, at least about .01 ⁇ M, at least about .05 ⁇ M, at least about 0.08 ⁇ M, at least about 0.09 ⁇ M, at least about 0.1 ⁇ M, at least about 0.2 ⁇ M, at least about 0.3 ⁇ M, at least about 0.4 ⁇ M, at least about 0.5 ⁇ M, at least about 0.6 ⁇ M, at least about 0.7 ⁇ M, at least about 0.8 ⁇ M, at least about 0.9 ⁇ M, at least about 1 ⁇ M, at least about 1.5 ⁇ M, at least about 2 ⁇ M, or about 2 ⁇ M to 10 ⁇ M compound of the present invention.
  • NF2-deficient or NFl -deficient tumor cells are contacted with a compound of the present invention in vivo.
  • a compound of the present invention in this embodiment, a compound of the
  • 53412 v2 DC 63 present invention is administered to a subject with a NF2-deficient or NFl -deficient tumor.
  • the subject can be any mammal, including, but not limited to, a human, canine, rat, mouse, and farm animals.
  • the compound can be administered at the site of the tumor or elsewhere in the body.
  • a compound of the present invention When a compound of the present invention is administered in vivo, it can be administered such that the serum levels mimic those levels found to cause inhibition of cell growth and/or cell death in vitro, In one embodiment, the compound of the present invention contacts the NF2-deficient or NFl -deficient tumor cells with another agent.
  • the present invention provides methods of treating a subject diagnosed with neurofibromatosis type-2 or a condition associated with the loss of NF2 function (i.e., the loss of the protein, Merlin), including, but not limited to sporadic schwannomas, and other NF2 related tumors, such as sporadic meningiomas and sporadic mesotheliomas.
  • the method comprises administering to a patient a therapeutically effective amount of one or more compounds of the present invention.
  • Subject' " and "'patient” are used interchangeably herein, and refer to a mammalian subject to be treated, including, but not limited to, human patients.
  • a subject can be diagnosed with NF2 by methods known in the art, including, but not limited to, a direct gene test and the bilateral occurrence of vestibular schwannomas.
  • the methods of treatment are similar for a subject diagnosed with NFl or a condition associated with the loss of NFl function (i.e., the loss of the protein, neurofibromin).
  • the present invention also provides methods of treating a subject diagnosed with a neurodegenerative disease.
  • a neurodegenerative disease is a central nervous system disorder characterized by gradual and progressive loss of neural tissue. Neurodegenerative diseases result from deterioration of neurons which over time will lead to neurodegeneration and disabilities resulting from this. Examples of neurodegenerative diseases include, but are not limited to Alzheimer ' s disease, amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease, Huntington disease, multiple sclerosis, Parkinson disease, and spinal muscular atrophy but other neurodegenerative diseases are known to persons skilled in the field.
  • ALS amyotrophic lateral sclerosis
  • Creutzfeldt-Jakob disease Huntington disease
  • multiple sclerosis Parkinson disease
  • spinal muscular atrophy but other neurodegenerative diseases are known to persons skilled in the field.
  • terapéuticaally effective amount refers to an amount sufficient to inhibit the growth of one or more NF-2 deficient tumors or NFl -deficient tumors, or to slow, stop, or reverse the gradual and progressive loss of neural tissue.
  • “Inhibition”, as used herein, means that the size of a tumor stabilizes and does not increase.
  • the tumor may be reduced in size following administration of a compound of the present invention. For instance, the tumor may be reduced in size at least about 10%, at least about
  • 53412 v2'DC 64 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the size of the tumor prior to treatment, i.e., a baseline measurement.
  • the inhibition of tumor growth can be assessed by methods known in the art, including, but not limited to, magnetic resonance imaging or a CAT scan.
  • a "therapeutically effective amount” will vary depending on the tumor load of the patient and the age, weight, and other conditions of the patient to be treated. Further, a therapeutically effective amount may vary based on the specific compound of the present invention administered and pharmaceutical composition as well as the route of administration.
  • the methods of the present invention can be used to treat a variety of NF2-deficient tumors or NFl -deficient tumors.
  • the compound of the present invention can be administered to treat a vestibular schwannoma, for instance, a unilateral schwannoma or a bilateral schwannoma. Inhibition of the growth of these tumors and/or death of tumor cells can mitigate the symptoms associated with schwannomas, including but not limited to vestibular schwannomas.
  • treatment with a compound of the present invention may result in an improvement in hearing, tinnitus and/or balance.
  • the methods of the invention can be used to treat other types of NF2-deficient schwannomas as well or type of NFl -deficient tumors.
  • administration of a compound of the present invention can be used to treat a patient suffering from one or more spinal cord schwannomas/neurofibromas and other peripheral nerve schwannomas/neurofibromas and sporadic schwannomas/neurofibromas.
  • Treatment of peripheral nerve tumors, including spinal cord tumors can decrease a patient ' s pain.
  • neurological deficits associated with NFl may be reversible with the treatment of the present invention.
  • administration of a compound of the present invention results in a decrease in a patient's tumor load.
  • administration of a a compound of the present invention can result in at least about a 5%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, at least about a 30%, at least about a 40%, at least about a 50%, at least about a 60%, at least about a 70%, at least about an 80%, at least about a 90%, or about a 100% decrease in tumor load.
  • the compound of the present invention may be formulated for administration in a pharmaceutically acceptable earner or excipient in accordance with known techniques in the
  • the compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the composition and must not be deleterious to the patient.
  • the earner may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for instance, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of compound.
  • the pharmaceutical composition administered to the patient contains more than one type of HSP90 inhibitor.
  • compositions include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, etc.), topical (e.g., both skin and mucosal surfaces, including airway surfaces) and transdermal administration.
  • the pharmaceutical composition is applied directly to the site of a NF2-deficient tumor or NFl -deficient tumor.
  • the HSP90 inhibitor can be applied by local treatment which encompasses both topical treatment and intralesional or intradermal treatment at the site of the tumor.
  • the inhibitor can be injected into, topically applied onto or near a NF2-deficient tumor or NFl-deficient tumor.
  • the inhibitor is applied intralesionally to NF2-deficient tumors or NF l -deficient tumors by methods known in the art.
  • the pharmaceutical composition can take the form of an implant. Such a composition can be surgically implanted at or near the site of a tumor for slow release of the compound of the present invention.
  • a pharmaceutical composition is administered by transcatheter arterial embolization by methods known in the art.
  • Pharmaceutical compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a compound of the present invention; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • compositions suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or
  • compositions of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
  • a pharmaceutical composition of the compound of the present invention is applied topically.
  • a pha ⁇ naceutical composition can be applied to the skin near a NF2-deficient tumor or a NFl -deficient tumor.
  • compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such compositions may be applied near or at the site of a NF2-deficient tumor or a NFl -deficient tumor.
  • Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.
  • Suitable formulations comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
  • the present invention also includes methods of treating a subject with NF2 or NFl by administering a liposomal formulation of a compound of the present invention.
  • the technology for forming liposomal suspensions is well known in the art.
  • the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome.
  • the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations containing the compound of the present invention disclosed herein or salts thereof may also be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • a pharmaceutically acceptable carrier such as water
  • compositions such as aqueous base emulsions, may be prepared from compounds of the present invention.
  • the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of compounds of the present invention.
  • Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.
  • the therapeutically effective dosage of any specific compound of the present invention for treatment of a NF2-deficient tumor or a NF 1 -deficient tumor will vary from patient to patient, and will depend upon factors such as the age, weight, gender and condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.01 or 0.1 to about 50, 100 or 500 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the compound, including the cases where a salt is employed.
  • Mammals, and specifically humans, suffering from a respiratory disorder can be treated by the inhalation, systemic, oral, topical, or transdermal administration of a composition comprising an effective amount of the compounds described herein or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier or diluent.
  • the compounds of the invention are in the form of an inhaled dosage.
  • the compounds may be in the form of an aerosol suspension, a dry powder or liquid particle form.
  • the compounds may be prepared for delivery as a nasal spray or in an inhaler, such as a metered dose inhaler.
  • Pressurized metered-dose inhalers (“MDF " ) generally deliver aerosolized particles suspended in chlorofluorocarbon propellants such as CFC-1 1, CFC- 12, or the non-chlorofluorocarbons or alternate propellants such as the fluorocarbons, HFC- 134A or HFC-227 with or without surfactants and suitable bridging agents.
  • Dry-powder inhalers can also be used, either breath activated or delivered by air or gas pressure such as the dry-powder inhaler disclosed in the Schering Corporation International Patent Application No. PCT/US92/05225, published 7 Jan. 1993 as well as the TurbuhalerTM (available from Astra Pharmaceutical Products, Inc.) or the RotahalerTM (available from Allen & Hanburys) which may be used to deliver the aerosolized particles as a finely milled powder in large aggregates either alone or in combination with some pharmaceutically acceptable earner e.g. lactose; and nebulizers.
  • the compounds of the invention may be also administered in specific, measured amounts in the form of an aqueous suspension by use of a pump spray bottle.
  • the aqueous suspension compositions of the present invention may be prepared by admixing the dry-powder inhaler disclosed in the Schering Corporation International Patent Application No. PCT/US92/05225, published 7 Jan. 1993 as well as the TurbuhalerTM (available from
  • the aqueous suspension compositions according to the present invention may contain, inter alia, water, auxiliaries and/or one or more of the excipients, such as: suspending agents, e.g., microcrystalline cellulose, sodium carboxymethylcellulose, hydroxpropyl-methyl cellulose; humectants, e.g. glycerin and propylene glycol; acids, bases or buffer substances for adjusting the pH, e.g., citric acid, sodium citrate, phosphoric acid, sodium phospate as well as mixtures of citrate and phosphate buffers; surfactants, e.g.
  • Typical systemic dosages for all of the herein described conditions are those ranging from 0.01 mg/kg to 1500 mg/kg of body weight per day as a single daily dose or divided daily doses. Preferred dosages for the described conditions range from 0.5-1500 mg per day. A more particularly preferred dosage for the desired conditions ranges from 5-750 mg per day.
  • Typical dosages can also range from 0.01 to 1500, 0.02 to 1000, 0.2 to 500, 0.02 to 200, 0.05 to 100, 0.05 to 50, 0.075 to 50, 0.1 to 50, 0.5 to 50, 1 to 50, 2 to 50, 5 to 50, 10 to 50, 25 to 50, 25 to 75, 25 to 100, 100 to 150, or 150 or more mg/kg/day, as a single daily dose or divided daily doses.
  • the compounds are given in doses of between about 1 to about 5, about 5 to about 10, about 10 to about 25 or about 25 to about 50 mg/kg.
  • Typical dosages for topical application are those ranging from 0.001 to 100% by weight of the active compound.
  • the compounds are conveniently administered in units of any suitable dosage form, including but not limited to one containing from about 7 to 3000 mg, from about 70 to 1400 mg, or from about 25 to 1000 mg of active ingredient per unit dosage form.
  • an oral dosage of from about 50 to 1000 mg is usually convenient, including in one or multiple dosage forms of 50, 100, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 mgs.
  • Lower dosages may be preferable, for example, from about 10-100 or 1 -50 mgs.
  • Also contemplated are doses of 0.1 -50 mg, 0.1 -20 mgs., or 0.1-10 mgs.
  • lower doses may be utilized in the case of administration by a non-oral route, as for example, by injection or inhalation.
  • the compound is administered for a sufficient time period to alleviate the undesired symptoms and the clinical signs associated with the condition being treated.
  • the active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutic amount of compound in vivo in the absence of serious toxic effects.
  • Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions are generally known in the art. They include, but
  • 53412 v2 DC 59 are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, solvents, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silicates, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, oils, polyemylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, solvents,
  • Pharmaceutically accepted vehicles can contain mixtures of more than one excipient in which the components and the ratios can be selected to optimize desired characteristics of the formulation including but not limited to shelf-life, stability, drug load, site of delivery, dissolution rate, self-emulsification, control of release rate and site of release, and metabolism.
  • Formulations can be prepared by a variety of techniques known in the art. Examples of formulation techniques can be found in literature publications and in texts such as "Water- insoluble drug formulation", edited by Rong Liu, 2000, Interpharm Press.
  • compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • a long-chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • a long-chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • surfactants such as Tweens, Spans and other surface- active emulsifying agents or bioavailability enhancers which are commonly used in the
  • the concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • Oral compositions will generally include an inert diluent or an edible earner. They may be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.
  • the compound or its salts can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable earners. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.
  • liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
  • appropriate lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol
  • Suitable vehicles or earners for topical application can be prepared by conventional techniques, such as lotions, suspensions, ointments, creams, gels, tinctures, sprays, powders, pastes, slow-release transdermal patches, suppositories for application to rectal, vaginal, nasal or oral mucosa.
  • thickening agents, emollients, and stabilizers can be used to prepare topical compositions.
  • thickening agents examples include petrolatum, beeswax, xanthan gum, or polyethylene, humectants such as sorbitol, emollients such as mineral oil, lanolin and its derivatives, or squalene.
  • the compound of present invention can also be mixed with other active agents which do not impair the desired action, or with materials that supplement the desired action.
  • the one or more active agents can be administered in conjunction, i.e. combination or alternation, with other medications used in the treatment of disorders or conditions associated with NFl or NF2.
  • active agent has the same meaning as described above.
  • methods of the invention include co-administering a compound of the present invention with another therapeutic agent, either concurrently or sequentially.
  • the compounds of the present invention can be administered in combination or alternation with one or more HSP90 inhibitors as described above.
  • a compound of the present invention can be administered with a cytotoxic agent known in the art to inhibit tumor growth or kill the tumor(s). Coadministration of a HSP90 pathway inhibitor with a cytotoxic agent may reduce the toxic side-effects otherwise associated with the cytotoxic agent. Toxicity would be reduced because less cytotoxic agent would need to be administered to the patient to inhibit tumor growth or kill one or more tumors than if the cytotoxic agent were administered alone. [00221] In addition to co-administration of the compound of the present invention with a cytotoxic agent such a chemotherapy drug, the compound of the present invention can also be administered with radiation therapy in combination.
  • the compound of the present invention can also be administered before or after surgical removal of a tumor to kill or inhibit the growth of any remaining NF2-deficient or NFl -deficient tumor cells.
  • This embodiment is especially useful for tumors of the brain and spine where removing all NF2-deficient or NFl -deficient tumor cells could put the patient at risk for permanent nerve damage or even death.
  • the compound of the present invention is administered to a patient with a PAK inhibitor.
  • any compound of the invention with one or more other active agents in a unitary dosage form for simultaneous or sequential administration to a patient.
  • the combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.
  • Co-administration of a compound of the invention with one or more other active agents generally refers to simultaneous or sequential administration of a compound of the invention and one or more other active agents, such that therapeutically effective amounts of the compound of the invention and one or more other active agents are both present in the body of the patient.
  • Co-administration includes administration of unit dosages of the compounds of the invention before or after administration of unit dosages of one or more other active agents, for example, administration of the compounds of the invention within seconds, minutes, or hours of the administration of one or more other active agents.
  • a unit dose of a compound of the invention can be administered first, followed within seconds or minutes by administration of a unit dose of one or more other active agents.
  • one or more other active agents can be administered first, followed by administration of a unit dose of a compound of the invention within seconds or minutes. In some cases, it may be desirable to administer a unit dose of a compound of the invention first, followed, after a period of hours (e.g., 1 -12 hours), by administration of a unit dose of one or more other active agents. In other cases, it may be desirable to administer a unit dose of one or more other active agents first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a compound of the invention.
  • the combination therapy may provide "synergy” and "synergistic effect", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect may be attained when the active ingredients are: (1 ) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills or capsules, or by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e.
  • the present invention also includes methods of screening HSP90 inhibiting compounds for efficacious treatment of NF2 or NFl .
  • the methods of the invention can be used to determine the efficacy of a compound in pre-clinical experiments. This is useful because it can allow researchers to reduce the number of drugs to be tested for efficacious treatment of NF2 or NFl .
  • the methods of the invention can also be used to repurpose existing drugs that were developed and/or are used for other indications.
  • efficacious treatment means that a test compound results in the inhibition or reduction of cell growth. In one embodiment, efficacious treatment results in cell death or inhibition of proliferation.
  • An efficacious treatment can be correlated with a reduction in the amount or activity of one or more client proteins of HSP90 or an increase in other HSP proteins such as HSP70. For instance, a reduction in the amount of ErbB2 protein leads to the reduction in the amount of phospho-ErbB2 and the activity of ErbB2 which can be correlated with the inhibition or reduction of growth of NF2-deficient cells.
  • a reduction in the amount of ErbB2 protein leads to the reduction in the amount of phospho-ErbB2 and the activity of ErbB2 which can be correlated with the inhibition or reduction of growth of NF2-deficient cells.
  • 53412 v2/ DC 74 reduction in the amount of AKT or phospho-AKT may lead to the reduction in the phosphorylation of mTOR, GSK3, FKHR, S6 and S6K which can also be correlated with the inhibition or reduction of growth of NF2-deficient cells or apoptosis of NF2-deficient cells.
  • a reduction in the amount of Raf may lead to the reduction in the activity of downstream MAP kinase signaling pathway which can be correlated with the inhibition or reduction of growth of NFl -deficient cells.
  • Methods known in the art can be used to assess whether a test drug reduces the activity of one or more client proteins of the HSP90 complex.
  • Such assays can be conducted in vitro, ex vitro using cells or tumor specimens from human patients or animal models, or, in the case of animal models, in vivo (Roe et al., 2004, Cell 1 16: 87-98; Smith et al., 2005,
  • the ability of a drug to reduce the activity of a client protein (e.g. a kinase) of HSP90 or the downstream pathway of the client protein is assessed by contacting the proteins of the cells with an anti-phospho antibody specific for the particular protein.
  • a client protein e.g. a kinase
  • Western blots can be used by methods known in the art and can be probed with anti-phospho antibodies to phosphorylated AKT, phosphorylated ErbB2, phosphorylated GSK3, phosphorylated S6, phosphorylated S6K, phosphorylated mTOR, phosphorylated Mek, and phosphorylated down-stream proteins thereof.
  • Embodiments of the invention are summarized as follows:
  • the present invention is directed to a method of treating, preventing or ameliorating tumors or symptoms resulting from neurofibromatosis in a subject comprising administering to said subject with neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or with neurofibromatosis type 1 (NFl) or a condition associated with the loss of NFl function a therapeutically effective amount of at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor which inhibits or slows growth of one or more NF2-deficient tumors or NFl -deficient tumors, reduces the number of said tumors or inhibits and/or reduces associated symptoms as compared to no treatment with the composition as a control level to determine treatment utility.
  • the method is particularly directed to the administration of the at least one composition comprising HSP90 resulting in a decrease in size and/or number of one or more NF2-deficient tumors or of one or more NFl- deficient tumors.
  • the method of the present invention comprises administering a HSP90 inhibitor comprising a compound which inhibits or reduces the function of HSP90 complex. More specifically, the HSP90 inhibitor comprises a compound that binds and inhibits the HSP90 protein, modifies HSP90 protein posttranslationally, and/or increases HSP70 or other HSP proteins from their normal levels in the NF2-deficient or NFl -deficient tumors. The administration of the HSP90 inhibitor results in the upregulation or increase in HSP70 in said subject. Further, the method comprises administering a HSP90 inhibitor comprising a compound that degrades or reduces one or more client proteins of HSP90 and the phosphorylated forms of the client proteins.
  • the method comprises administering a HSP90 inhibitor comprises a compound that inhibits or reduces activity or phosphorylation of signaling pathway proteins associated with one or more client proteins of HSP90.
  • the one or more client proteins are selected from the group consisting of ErbB2, AKT, and Raf.
  • the HSP90 inhibitor inhibits or reduces activity or phosphorylation of the signaling pathways proteins, such as PDK, mTOR, GSK3, 4E-BP1 , Bad, FKHR, HSP90, S6K, S6, Mek, and Erkl/2.
  • the method comprises administering a HSP90 inhibitor comprising a biological agent or molecule that is a peptide, antibody, siRNA and antisense nucleic acid molecule.
  • the method comprises administering at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor comprising radicicol or a derivative thereof, 17-AAG or a derivative thereof, a purine scaffold-based HSP90 inhibitor or a derivative thereof, a pyrazole scaffold-based HSP90 inhibitor or a derivative thereof, an imidazole scaffold-based HSP90 inhibitor or a derivative thereof, a tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitor or a derivative thereof, Novobiocin or a derivative thereof, or any combination thereof.
  • HSP90 heat shock protein 90
  • the HSP90 inhibitor comprises a HSP90 modulator compound such as a HDAC inhibitor and specifically HDAC6 inhibitor.
  • This HSP90 modulator compound may further comprise Trichostatin A, SAHA, LAQ824, FK228, or derivatives thereof.
  • the method comprises administering a HSP90 inhibitor comprising an inhibitor or compound that modulates or increases the level of HSPs such as HSP70 and HSP27.
  • the HSP90 inhibitor further comprises NZ28 (NCS-134754) or a derivative thereof.
  • the method comprises administering the HSP90 inhibitor orally, intravenously or locally, such as intralesionally or topically.
  • the method also encompasses administering the HSP90 inhibitor before, during or after surgical removal of a tumor, Another mode of
  • the method of the present invention treats, prevents or ameliorates tumors or symptoms resulting from neurofibromatosis type 2 (NF2) or condition(s) associated with the loss of NF2 function. More specifically the one or more NF2-deficient tumors comprise vestibular schwannomas, and more specifically comprise a unilateral vestibular schwannoma or a bilateral vestibular schwannoma.
  • NF2 neurofibromatosis type 2
  • the one or more NF2-deficient tumors that are treated comprise spinal cord schwannomas, sporadic schwannomas, peripheral nerve schwannomas, schwannoma, meningioma, mesothelioma, ependymoma, glioma and astrocytoma.
  • the method comprises the administration of the HSP90 inhibitor to obtain results in an improvement in at least one of the subject's hearing, balance and vision; increase in muscle mass, reduction in tumor burden in the subject, which the latter is identified using a MRI or a CAT scan.
  • the method of the present invention treats, prevents or ameliorates tumors or symptoms resulting from neurofibromatosis type 1 (NFl) or condition(s) associated with the loss of NFl function.
  • NFl neurofibromatosis type 1
  • the one or more NFl -deficient tumors comprise a dermal and plexiform neurofibromas, optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ependymomas, pheochromocytomas and ganglioneuromas, rhabdomyosarcomas, neurofibrosarcomas, malignant peripheral nerve sheath tumors ("MPNST”), malignant schwannomas, and JMML.
  • MPNST peripheral nerve sheath tumors
  • the present invention also includes a method of inhibiting or reducing the growth or number of NF2-deficient tumor cells or NFl -deficient tumor cells comprising contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor which inhibits or slows growth and/or reduces the number of one or more NF2-deficient tumors or NFl -deficient tumors.
  • This method comprises contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with said compound occurs in vitro or ex vivo.
  • the NF2-deficient tumor cells are Nf2-deficient mouse Schwann cells and said NFl -deficient tumor cells are Nf 1 -deficient mouse Schwann cells.
  • the NF2-deficient tumor cells are NF2-deficient human schwannoma cells and said NFl -deficient tumor cells are NFl -deficient human Schwann cells.
  • the NF2-deficient tumor cells are selected from the group consisting of NF2-deficient schwannoma cell line cells, NF2-deficient meningioma cell line cells and NF2-deficient mesothelioma cell line cells.
  • the NF2-deficient tumor cells are selected from the group
  • the NFl -deficient tumor cells are selected from the group consisting of human MPNST cells, primary neurofibroma cells derived from NFl patients, mouse N/Z/pJJ-deficient MPNST cell lines established from cisNfl ;p53 mice, and NfI-/- mouse cells, such as Schwann cells, mouse embryonic cells, and leukemia cells. More specifically, the NFl -deficient tumor cells are selected from the group consisting of ST88-14, 88-3, 90-8, and sNF96.2.
  • the NF2-deficient tumor cells or NFl- deficient tumor cells that are contacted with said HSP90 inhibitor can occur in vivo.
  • the NF2-deficient tumor cells or said NFl -deficient tumor cells are from a human, canine, rat or mouse.
  • the method of the present invention comprises contacting the NF2-deficient tumor cells or the NFl -deficient tumor cells with a HSP90 inhibitor that results in an inhibition of HSP90 function.
  • the method further comprises contacting the NF2-deficient tumor cells or said NFl -deficient tumor cells with said HSP90 inhibitor that results in an upregulation of HSP70.
  • the contact of the NF2-deficient tumor cells or the NFl -deficient tumor cells with the HSP90 inhibitor results in degradation of ErbB2 and/or phosphorylated ErbB2, in degradation of Akt and/or phosphorylated Akt or in degradation of Raf and/or phosphorylated Raf. More specifically, the contact of the NF2-deficient tumor cells or said NFl -deficient tumor cells with the HSP90 inhibitor results in a reduction in phosphorylation of proteins downstream of the ErbB2, Akt or Raf signaling pathway. The degradation or upregulation of the proteins or reduction in phosphorylated proteins is detected using an antibody.
  • the method also contacts a HSP90 inhibitor with the NFl -deficient and NF2- deficient cells where the HSP90 inhibitor comprises a biological agent or molecule, wherein the biological agent or molecule is selected from the group consisting of peptide, antibody, siRNA and antisense nucleic acid.
  • the method comprises contacting the NF2-deficent tumor and NFl -deficient tumors with at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor comprising radicicol or a derivative thereof, 17-AAG or a derivative thereof, a purine scaffold-based HSP90 inhibitor or a derivative thereof, a pyrazole scaffold-based HSP90 inhibitor or a derivative thereof, an imidazole scaffold-based HSP90 inhibitor or a derivative thereof, a tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitor or a derivative thereof, Novobiocin or a derivative thereof, or any combination thereof.
  • the HSP90 inhibitor comprises a HSP90 modulator compound such as a HDAC inhibitor and specifically HDAC6 inhibitor. This HSP90 modulator compound may further comprise Trichostatin A, SAHA, LAQ824, FK228,
  • the method comprises administering a HSP90 inhibitor comprising an inhibitor or compound that modulates or increases the level of HSPs such as HSP70 and HSP27.
  • the HSP90 inhibitor further comprises NZ28 (NCS-134754) or a derivative thereof.
  • the HSP90 inhibitor also comprises an inhibitor or compound that modulates (e.g. increases) the level of HSPs such as HSP70 and HSP27.
  • the HSP90 inhibitor may also comprise NZ28 (NCS- 134754) or a derivative thereof.
  • the method also comprises contacting the NF-2-deficient cells or NF-I -deficient cells are contacted with said compound and a compound that inhibits or reduces PAK activity.
  • the present invention includes a method for screening a test compound for treatment of NF-2 or NF-I comprising treating or contacting NF-2-deficient cells or NF-I -deficient cells with said test compound, wherein a degradation of one or more client proteins of HSP90 or a decrease in activity of signaling pathways associated with one or more client proteins of HSP90 or an increase in HSP70 is indicative of an efficacious treatment of NF-2 or NF-I .
  • the method further comprises assessing inhibition of HSP90 function.
  • the inhibition of HSP90 function results in an upregulation of HSP70.
  • the one or more client proteins of HSP90 are selected from the group consisting of ErbB2, AKT, and Raf.
  • the signaling pathways are associated with one or more client proteins of HSP90 are ErbB2 pathway, AKT pathway or Raf pathway which contain at least one protein selected from group consisting of ErbB2, AKT, Raf, mTOR, GSK3, 4E-BP1 , Bad, FKHR, S6K, S6, Mek, and Erkl/2.
  • the one or more client proteins is AKT, the AKT is degraded by said test compound, resulting in reduced phosphorylation of AKT.
  • the treatment results in reduced phosphorylation of S6, GSK3, FKHR, Mek, or Erkl/2.
  • the reduced phosphorylation is detected using an antibody.
  • the method of the present invention further comprises measuring NF-2-deficient cells or NF- 1 -deficient cells following treatment with the test compound, wherein a decrease in the number of NF2-deficient cells or NF-I -deficient cells following treatment with the test compound or a decrease in proliferation of NF2-deficient cells or NF-I -deficient cells following treatment with the test compound is indicative of an efficacious treatment.
  • the method further comprises comparing the NF2-deficient cells or NF-I- deficient cells following treatment to untreated NF2 deficient cells or NF-I -deficient cells, wherein a decrease in one or more client proteins of the HSP90 following treatment with the test compound compared to untreated NF2-deficient cells or NF-I -deficient cells is indicative of an efficacious treatment. Additionally, the method further comprising comparing the NF2-deficient cells or NF-I -deficient cells following treatment to untreated NF2 deficient
  • NF2-deficient cells or NFl -deficient cells wherein a decrease in number of NF2 deficient cells or NF-I- deficient cells following treatment with the test compound or a decrease in proliferation of NF2-deficient cells or NF-I -deficient cells following treatment with the test compound compared to untreated NF2-deficient cells or NF-I -deficient cells is indicative of an efficacious treatment.
  • the treatment of the NF2-deficient cells or NFl -deficient cells with said test compound occurs in vitro or ex vivo.
  • the NF2-deficient tumor cells are Nf2- deficient mouse Schwann cells and the NFl -deficient tumor cells are Nf 1 -deficient mouse Schwann cells.
  • the NF2-deficient tumor cells are NF2-deficient human schwannoma cells and said NFl -deficient tumor cells are NFl-deficient human Schwann cells.
  • the NF2-deficient tumor cells are selected from the group consisting of NF2- deficient schwannoma cell line cells, NF2-deficient meningioma cell line cells and NF2- deficient mesothelioma cell line cells.
  • the NF2-deficient tumor cells are selected from the group consisting of HEI193 cells, SF1335 cells, BAR cells and RAV cells.
  • the NFl-deficient tumor cells are selected from the group consisting of human MPNST cells, primary neurofibroma cells derived from NFl patients, mouse iV/7,7?53-deficient MPNST cell lines established from cisN/I;p53 mice, and NfJ-/- mouse cells, such as Schwann cells, mouse embryonic cells, and leukemia cells. More specifically, the NFl -deficient tumor cells are selected from the group consisting of ST88-14, 88-3, 90-8, and sNF96.2.
  • the NF2-deficient tumor cells or NFl -deficient tumor cells that are contacted with said HSP90 inhibitor can occur in vivo.
  • the NF2-deficient tumor cells or said NFl-deficient tumor cells are from a human, canine, rat or mouse. Processes for the Preparation of the Compounds
  • LiHMDS Lithium hexamethyldisilazide LiN(SiMe 3 ⁇ ) ⁇ M Micromolar concentration ( ⁇ mol.l "1 )
  • PS-TBD ( 1 ,5,7)-Triaza-bicyclo[4.4.0]dodeca-5-ene-7-methyl polystyrene
  • Retrosynthetic disconnections for pochonin D (2-85) depicting the synthetic strategy are shown below.
  • EOM protecting groups were chosen for phenolic protection.
  • mono-EOM chlorinated acid 2-108 was synthesized in three steps from formylated orcinol 2-94a (Scheme 7).
  • the selective deprotection of the ortho-phenol was achieved using a specific concentration of TFA in a THF/MeOH mixture (THF/TFA/MeOH 7: 1.5: 1 (vol.)) without any bis-deprotection.
  • Further Mitsunobu esterification using standard protocol (DIAD, PPh 3 , toluene) and reprotection of the ort/io-phenol allowed the formation of compound 2-110 in 51 % yield from acid 2-108.
  • pochonin A (2-122) was then prepared not only to confirm its structure but also to compare its biological activity to pochonin D and radicicol.
  • Epoxidation of pochonin D using DMDO allowed the formation of pochonin A as a 1 : 1 mixture of diastereoisomers that could be separated by column chromatography. Attempts to improve the selectivity of this reaction using epoxone ((a) Tu, Y. et al, J Am Chem Soc 118, 9806-9807 (1996), (b) Wang, Z.-X. et al., J Org Chem 62, 2328-2329 (1997)) as a chiral auxiliary failed completely with no conversion at 0 0 C or at room temperature. The lack of solubility of pochonin D at low temperature did not allow for improvement of this selectivity and other routes were pusued. As depicted in Scheme 1 1, the epoxidation of the bis-EOM
  • Scheme 11 Direct conversion of bis-EOM pochonin D (2-112) a) DMDO (1.0 equiv.), CH 3 CN, O 0 C ⁇ 23 °C. 1.5 h , 79 %; b) PS-TsOH (10.0 equiv., 3.2 mmol.g " '), MeOH, 40 0 C, 1 h.
  • Weinreib amide 2-114 (1.0 equiv.), 10 min, 60 %; d) Grubbs' II (10 % mol), toluene (2 mM), 80 0 C. 12 h, 87 %; e) DMDO (1.0 equiv.), CH 3 CN, 0 ⁇ 23 0 C, 1.5 h, 83 %, 1 : 1 mixture of diastereoisomers; f) MgBr 2 -Et 2 O (8.0 equiv.), CH 2 Cl 2 , 23 0 C, 1 h. 70 %.
  • homoallylic alcohols 2-134 bearing various substituent at R 2 were envisioned. Homoallylic alcohols 2-134 that are not commercially available may be obtained in by any suitable method. In one embodiment, the homoallylic alcohols 2-134 were obtained in their highest enantiomeric form either by enzymatic resolution 2 of the racemic alcohol or by means of Brown allylation 168 of the corresponding aldehyde (H. E.Master et ⁇ l., Tet. Lett,, 37:9253 (1996); S. Singh et ⁇ l., Tet. Asymm., 13:2679 (2002) or via Brown allylation of the corresponding aldehyde. (H. C. Brown and P. K. Jadhav J. Am.
  • Macrocycles 2-112a-g and 2-120a-g were then used as the starting point for further diversifications.
  • Deprotection of the EOM groups of 2-112a-g and 2-120a-g using sulfonic acid resin afforded compounds 2-103a-g and 2-85a-g in pure form and excellent yields after simple filtration of the resin and evaporation of the solvents (Scheme 17).
  • the 12-membered ring products 2-121a-g were deprotected just as smoothly (not shown).
  • EOM deprotection under acid catalysis were slower for the chlorinated analogues and had to be monitored as prolonging the reaction times could lead to conjugate additions.
  • ketones 2-112a- g and 2-120a-g could be reduced using BER-resin to obtain both diastereoisomers of 2-14Ia- g in -60% yield.
  • Deprotection of the EOMs with sulfonic acid resin under regular conditions afforded compounds 2-142a-g.
  • Acetylation of the reduced intermediates 2-141a-g using PS- NMM/ Ac 2 O yielded compounds 2-143a-g which led to elimination upon deprotection to afford trienes 2-144 as a mixture of olefin geometries.
  • BER resin borohydride exchange resin
  • PS-TsOH sulfonic acid resin MP
  • DIBAL diisobutylaluminum hydride.
  • DMAP dimethylaminopyridine
  • DMF dimethylformamide
  • PS-NMM morpholinomethyl polystyrene.
  • R 3 X EOMCI, BrCH 2 COOfBu
  • AIlOH Allylalcohol
  • DMDO dimethyldioxirane
  • NMO 4-methylmor ⁇ holine iV-oxide
  • PS-DEAD ethoxycarbonylazocarboxymethyl polystyrene
  • PS-TBD TBD-methyl polystyrene
  • PS-TMABH 3 CN ( ⁇ olystirylmethyl)trimethylammonium cyanoborohydride.
  • PS-TsOH (1.0 equiv., 3.2 mmol.g 1 ), DHP (1.0 equiv.), CH 2 Cl 2 , 23 0 C, 5 h, -80 %: d) R 2 ONH 2 .HC1 (5.0 equiv.), Pyr/AcOH 5: 1 , 40 0 C, 12 h, -90 %; e) PS-TsOH (10.0 equiv., 3.2 mmol.g "1 ), MeOH, 40 0 C, 4 h, -80 % f) PS-TsOH (cat., 3.2 mmol.g "1 ), DHP (1.0 equiv.), CH 2 Cl 2 .
  • the bis-methylated compounds 2-164 were prepared (Scheme 22). Acid 2-108 was used in a standard Mitsunobu esterification with alcohol 2- 159. Compound 2-160 was protected as the ort/z ⁇ -phenol followed by acylation reaction with Weinreb amide 2-114 to yield the acyclic precursor 2-162. Ring-closing metathesis followed by removal of the EOM protecting groups on compound 2-163 using sulfonic acid resin furnished the bis-methylated analog 2-164.
  • oxime derivatives 2-165 and 2-167 were synthesized from macrocycle 2-163 as a separable mixture with the 1 ,4-addition product (Scheme 23). The carboxylic acid moiety of oxime 2-167 was then esterified to form the corresponding piperidine amide oxime 2-168. 94 Removal of the EOM groups using sulfonic acid resin allowed the isolation of both oximes 2-166 and 2-167 from 2-165 and 2-168 respectively. These two compounds along with their parent compound 2-164 and the 1 13 compounds from the library ( Figure 39) were then tested in biological assays for treating NFl and NF2.
  • Scheme 23 Synthesis of oxime derivatives of compound 2-164 a) BnONH 2 HCl or NH 2 OCH 2 CO 2 H (5 0 equiv.), Pyr/AcOH 5: 1 , 40 0 C, 24 h, 20-35 %, b) PS-TsOH (10 0 equiv., 3 2 mmol.g 1 ), MeOH, 40 0 C, 2.5 h, 77-80 %: c) P ⁇ e ⁇ dme (1.1 equiv ), EDC (1.1 equiv ), HOBt (1.1 equiv ), DMF, 23 0 C, 2 h, 75-80 %.
  • the pochonin oximes were prepared from cyclization of the pre-formed oximes as shown in Scheme 24.
  • protected pre-formed oxime 1 was bound to Wang resin using yV.yV'-diisopiOpylcarbodiimide and dimethylaminopyridine.
  • the allyl protecting group was removed and the phenol was functionalized under Mitsonubo conditions. This was followed by deprotection of the carboxylic acid and esterification with R 3 OH. Ring-closing was effected with the use of Grubbs II catalyst as described before to provide resin-bound oxime 5. Deprotection and removal from the resin was achived with trifluoroacetic acid to provide oxime 6.
  • the carboxylic acid can be reacted with a variety of groups R 4 XH to provide the oximes 7, wherein X is oxygen, sulfur, amino or substituted amino.
  • the oximes 7 were generally obtained as a 1 :1 mixture of E:Z isomers which could be separated by reverse-phase chromatograpy (C 18).
  • the pochonin oximes were prepared by an alternate process which utilized a Mitsunobu cyclization to construct the macrocylic ring.
  • Scheme 25 shows a non-limiting example for the formation of the macrocyle 2-al via a Mitsunobu esterification.
  • Orcinol (compound 8) is oxidized with phosphoryl chloride in DMF to provide aldehyde 9, which is protected as the di-ethoxyrnethyl ether and subjected to oxidative chlorination with NaClO 2 to produce carboxylic acid 10.
  • the carboxylic acid is protected as a trimethylsilylethyl ester and treated with LDA and Weinreb amide 12 (Scheme 26) to provide the ⁇ , ⁇ -unsaturated ketone 13.
  • Ketone 13 is reacted with carboxymethoxylamine hemihydrochloride in pyridine at 40° C to produce the corresponding oximes as a mixture of E and Z isomers.
  • the oximes were converted to the desired amides 14 as a mixture of E and Z isomers by treatment with EDC and piperidine. Macrocyclization of precursor 14 to protected compound 15 was performed under Mitsunobu conditios by slow addition of DIAD to a solution of compound 14 and PhP 3 in toluene.
  • the phenol groups were deprotected by treatment with sulfonic acid resin at 40° C to produce 2a-l as a mixture of E and Z isomers.
  • the mixture of E- and Z-isomers was separated to obtain the pure E and Z isomers 2a-l.
  • the diene Weireb amide 12 was prepared according to the process shown in Scheme 26. Trans-3-hexenedioic acid dimethyl ester 16 was reduced to the corresponding diolwith lithium aluminum hydride. The diol was mono-protected as the tert-butyldiphenylsilyl ether 17, and the free alcohol was converted to aldehyde 20 in three steps via the nitrile 19. Aldehyde 20 was then treated with Weireb amide ylide 21 to produce the diene Weireb amide 12, which was used to prepare compound 13 (Scheme 24).
  • the solubilityof compounds 2a- 1 and 2a- 13 was determined. Both compounds were determined to be highly soluble in DMSO and DMA (> 5 g/mL). The good solubility of the compounds in DMSO and DMA enable formulations for intravenous or intraperitoneal administration. In one non-limiting example, a formulation of the oximes in DMSO/Tween 20/0.9% NaCl (10/5/85) was prepared. Biological Activity
  • IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer.
  • LC-MS were recorded using an Agilent 1 100 ⁇ HPLC with a Bruker' 1 micro-TOF instrument (ESI). Unless otherwise stated, a Supelco 15 C8 (5 cm x 4.6mm, 5 ⁇ m particles) column was used with a linear elution gradient from 100% H 2 O (0.5% HCO 2 H) to 100% MeCN in 13 min at a flow rate of 0.5 ml/min.
  • EXAMPLE 4 General procedure for the EOM deprotection to generate compounds deprotected-2-121 and 2-85.
  • EXAMPLE 5 General procedure for the synthesis of compounds 2-141.
  • Scheme 17 To a solution of corresponding compound 2-120/2-112 (1.0 equiv) in MeOH (0.03 M) at 0 0 C was added BER-resin (Borohydride on Amberlite*, 1.0 equiv, 2.5 mmol g " 1 ) and the reaction was stirred over 12 h. The reaction was then filtered and concentrated under reduced pressure. Purification by flash chromatography (SiO?, 0-20% EtOAc/cyclohexane gradient) afforded 2-141 (-60%) as a mixture of two diastereoisomers (1 : 1 ).
  • An illustrative example of compound 2-141 follows; its characterization data are in the text below the figure.
  • EXAMPLE 7 General procedure for the synthesis of compounds 2-143. [00305] As depicted in Scheme 17, to a solution of the corresponding compound 2-141 (1.0 equiv) in DMF (0.02 M) were added Ac 2 O (1.2 equiv), morpholinomethyl polystyrene (1.2 equiv, 3.2 mmol g "1 ) and DMAP (0.05 equiv) at 23 0 C and the mixture was stirred for 30 min, followed by TLC until consumption of the starting material. Then, the resin was filtered and the organic phase was concentrated under reduced pressure.
  • EXAMPLE 8 General procedure for the synthesis of compounds 2-144.
  • Scheme 17 To a solution of corresponding compound 2-143 (1.0 equiv) in MeOH (0,02 M) was added PS-TsOH (10.0 equiv, 3.2 mmol/g) and the suspension was shaken at 40 0 C for 4 h. The reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. Purification by PTLC (SiO 2 , 20% EtOAc/cyclohexane) afforded compounds 2-144 (-60% yield).
  • PTLC SiO 2 , 20% EtOAc/cyclohexane
  • EXAMPLE 10 General procedure for the synthesis of compounds 2-146. [00308] As depicted in Scheme 19, to a solution of corresponding compound 2-103/2-85 (1.0 equiv) in CH 2 Cl 2 /Ac0H 10/1 (0.08 M) (polystyrylmethyl)trimethylamrnonium cyanoborohydride (2.0 equiv, 3.5 mmol g *1 ) was added at 23 0 C and the reaction was monitored by TLC until the starting material had been consumed (4 h). Then, the resin was filtered and the organic phase was concentrated under reduced pressure. Purification by PTLC (SiO 2 , 30% EtOAc/cyclohexane) afforded compounds 2-146 (50-60%). An illustrative example of compound 2-146follows; its characterization data are in the text below the figure.
  • EXAMPLE 1 1 General procedure for the synthesis of compounds 2-147. [00309] As depicted in Scheme 19, to a solution of corresponding compound 2-103/2-85 (1.0 equiv), in THF (0.05 M) were added in a sequential manner the corresponding alcohol (2.0 equiv), triphenylphosphine (2.0 equiv) and ethoxycarbonylazocarboxymethyl polystyrene (2.0 equiv, 1.3 mmol g "1 ).
  • EXAMPLE 13 General procedure for the synthesis of compounds 2-149. [00311] As depicted in Scheme 19, to a solution of compound 2-103/2-85 (1.0 equiv) in acetone/H 2 0 10/1 (0.05 M) was added OsO 4 (0.1 equiv) followed by NMO (1.0 equiv) at 23 0 C and the mixture was stirred for 1 h. The crude mixture was filtered through a plug of silica,
  • EXAMPLE 14 General procedure for the synthesis of compounds 2-150.
  • Scheme 19 To a solution of compound 2-103/2-85 (1.0 equiv) in CH 3 CN (0.03 M) at 0 0 C was added freshly made DMDO (1.2 equiv, 0.04 M in acetone) and the mixture was stirred for 30 min. After evaporation of the solvents under reduced pressure, purification by PTLC (SiO 2 , 30% EtOAc/cyclohexane) afforded epoxides 2-150 (>90%) as a mixture of two diastereoisomers.
  • PTLC SiO 2 , 30% EtOAc/cyclohexane
  • EXAMPLE 15 General procedure for the synthesis of compounds 2-151. [00313] As depicted in Scheme 20, to a solution of compound 2-120 (1.0 equiv) in dioxane (0.05 M) at 23 0 C was added HCl COnc. (20 equiv), and the mixture was stirred for 3 h. After that time the reaction was filtered through a plug of silica gel, the solvents were evaporated under reduced pressure, and purified by PTLC (SiO 2 , 30% EtOAc/cyclohexane) to afford compound 2-151 (>75%) as a mixture of two diastereoisomers. Illustrative examples of compound 2-151 follow; their characterization data are presented respectively in the text below each figure.
  • EXAMPLE 16 General procedure for the elimination of ⁇ -Cl from compounds 2-151.
  • EXAMPLE 18 General procedure for the synthesis of compounds 2-154, [00316] As depicted in Scheme 20, to a solution of corresponding compound 2-120 (1.0 equiv) in pyridine/AcOH (5/1 , 0.03 M) was added the corresponding hydroxylamine (5.0 equiv) and the mixture was heated up to 40 0 C. After stirring overnight the solvents were evaporated under reduced pressure with SiO 2 . Elution of the compound over a short path of SiO 2 with a mixture of 30% EtOAc/cyclohexane afforded after evaporation 2-154 (-99%) as a mixture of two diastereoisomers cis/trans). An illustrative example of compound 2-154 follows; its characterization data are in the text below the figure. Illustrative Compound 2-154:
  • EXAMPLE 19 General procedure for the synthesis of compounds 2-155. [00317] As depicted in Scheme 20, to a solution of compound 2-154 (1.0 equiv) in MeOH (0.02 M) was added PS-TsOH (10.0 equiv, 3.2 mmol/g) and the suspension was shaken at 40 0 C for 4 h. The reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. The crude product obtained was submitted without further purification to the next step. Thus to a solution in CH 2 Cl 2 (0.02 M ) of this crude at 23 0 C were added DHP (1.0 equiv) and PS-TsOH (cat, 3.2 mmol/g) and the mixture was stirred for 5 hours.
  • EXAMPLE 21 General procedure for the synthesis of compounds 2-157. [00319] As depicted in Scheme 21, to a solution of compound 2-128 (1.0 equiv) in pyridine/AcOH (5/1 , 250 ⁇ L) was added the corresponding hydroxylamine (5.0 equiv) and the mixture was heated up to 40 0 C. After stirring overnight the solvents were evaporated under reduced pressure, and filtration on SiO 2 with a mixture of 30% EtOAc/cyclohexane afforded after evaporation two isomers of 2-157 ⁇ 90%.
  • An illustrative example of compound 2-157 follows; its characterization data are in the text below the figure.
  • EXAMPLE 22 General procedure for the synthesis of compounds 2-158. [00320] As depicted in Scheme 21 , to a solution of corresponding compound 2-157 (1.0 equiv) in THF was added TBAF (2.5 equiv, I M solution in THF) and the mixture was stirred at room temperature for 2 hours. The solvents were then evaporated under reduced pressure, and filtration on SiOo with a mixture of 30% EtOAc/cyclohexane afforded after evaporation, compounds 2-158 in >85 % yield. An illustrative example of compound 2-158 follows; its characterization data are in the text below the figure. Illustrative Compound 2-158;
  • EXAMPLE 23 General procedure for the synthesis of compounds 2a- 1 via Mitsunobu cyclization.
  • the present invention includes assaying compounds of the present invention, i.e., test compounds, for use as therapeutics for NF2 or NFl .
  • test compounds for use as therapeutics for NF2 or NFl .
  • three compounds namely NXD30001 , NXD30002, and NXD3OOO3, are described below as representative compounds of the present invention.
  • NXD30001 is also referred to as E-2al in Table 1 of this application
  • NXD30002 is also referred to as Z-2-al in Table 1 of this application
  • NXD30017 is also referred to as 2-al3 in Table 1 of this application.
  • 45 deficient or NFl -deficient cells lines as well as NF2-deficient cells or NFl -deficient cells obtained from a patient with NF2 or NFl , respectively, are treated with varying amounts of test compounds, and the effects of the test compound on degradation of one or more client proteins of HSP90, on HSP70 upregulation, and on activity or phosphorylation status of proteins in pathways associated with one or more client proteins of HSP90 are measured.
  • Compounds that cause a decrease in the amount and/or activity, e.g. decrease or increase in phosphorylation, of one or more client proteins of HSP90, an upregulation of HSP70, or a decrease in activity, e.g. decrease or increase in phosphorylation, of proteins in pathways associated with one or more client proteins of HSP90 can be identified as a putative therapeutic for NF2 or NF 1.
  • the client proteins of HSP90 are involved in many signaling pathways that control diverse cellular functions such as cell growth and proliferation, differentiation, and apoptosis.
  • HSP90 inhibitor e.g. ErbB2, Akt, c-Raf, Cdk4, and etc
  • HSP90 inhibitor in order to determine proteins degraded and downstream pathways blocked by a HSP90 inhibitor in Nf2-/- Schwann cells or NfI-/- Schwann cells, one or more client proteins and downstream proteins such as ErbB2, Akt, c-Raf, Cdk4, mTOR, S6K, S6, GSK3, Mek, Erkl/2, Cyclin D can be monitored.
  • the effect of a HSP90 inhibitor on ErbB2 is assessed by comparing the amounts of ErbB2, phospho-ErbB2, and several phosphorylated downstream proteins (e.g. phospho-Akt, phospho-S6K, phospho-S6, and phospho-GSK3) in cells treated with the compound and those treated with vehicle (control).
  • phospho-Akt phospho-Akt
  • phospho-S6K phospho-S6K
  • phospho-GSK3 phospho-GSK3
  • Cells that are useful for treatment include, but are not limited to, NF2-deficient Schwann cells ⁇ e.g. HEI-193), NF2-deficient malignant mesothelioma cells (e.g. BAR and RAV), NF2-deficient meningioma cells ⁇ e.g. SF 1335), Nf2-deficient mouse cells (e.g. Schwann cells and mouse embryonic fibroblasts), and NF2-deficient schwannoma cells derived from schwannomas in a patient.
  • HSP90 inhibitors can be evaluated in a panel of NF2 mutant human and mouse cells and cell lines.
  • Antibodies to one or more client proteins of HSP90 e.g.
  • Cells that are useful for treatment include, but are not limited to, NFl -deficient human MPNST cells ⁇ e.g. ST88-14, 88-3, 90-8, and sNF96.2) (Basu et al., 1992, Nature 356: 713-715; DeClue et al., 1992, Cell 69: 265-273; Wallace et al., 2000, Genes Chromosomes Cancer 27(2): 1 17-123; Muir et al., 2001 , Am. J. Pathol. 158: 501-513), primary neurofibroma cells derived from NFl patients, mouse /V/7/pJ.?-deficient MPNST cell lines
  • NfI-/- mouse cells e.g. Schwann cells, mouse embryonic cells, and leukemia cells.
  • NXD30001 downregulates Akt, ErbB2, and c-Raf in NF2-def ⁇ cient mouse and human tumor cells
  • NXD30001 causes upregulation ofHSP70 in NF2-def ⁇ cient mouse and human tumor cells
  • NXD30001 downregulates EGFR, At, c-Raf, and Cdk4 in NF 1 -deficient human MPNST cells
  • Ras-Raf-Mek-Erk 1/2 pathway is activated due to the loss of NFl in these cells.
  • EGFR is overexpressed in a number of NFl -deficient human and mouse cells.
  • Human malignant peripheral nerve sheath tumor (MPNST) cell ST88-14 was treated with increasing concentration (8-1000 nM) of NXD30001 for 24 hr. Cells were lysed and clarified lysates were subjected SDS-PAGE, followed by immunoblot analysis using indicated antibodies.
  • Figure 2 demonstrates NXD30001 induced degradation of HSP90 client proteins EGFR, Akt, and c-Raf, as well as a cell-cycle regulating kinase Cdk4 with an IC50 of approximate 100 nM after 24 hr treatment. Equal lysate protein loading is shown by actin immunoblot.
  • NXDM)OOl, NXD30002, and NXD30017 inhibit proliferation of NFl- and NF2-deficient human and mouse cells
  • Inhibitors of HSP90 function is assayed for the ability to decrease the number of NF2 deficient tumor cells in a tumor, i.e., shrink a tumor, or reduce the proliferation of NF2 deficient tumor cells.
  • the ability of inhibitors to decrease the number of NF2 deficient tumor cells in a sample or reduce proliferation of NF2 deficient tumor cells is assessed using a variety of methods known in the art.
  • a small library of compounds of the present invention total 14 oxime compounds
  • Cells used are Nfi-/- mouse embryonic stem cell (MESC), NF2-deficient human SF1335 meningioma cells, NF2-deficient HEI193 schwanoma cells, NF2-deficient BAR and RAV mesothelioma cells, NF2-plus BLA mesothelioma cells, and NFl -deficient human malignant peripheral nerve sheath tumor (MPNST) cells.
  • EPC Nfi-/- mouse embryonic stem cell
  • NF2-deficient human SF1335 meningioma cells NF2-deficient HEI193 schwanoma cells
  • BAR and RAV mesothelioma cells NF2-plus BLA mesothelioma cells
  • NFl -deficient human malignant peripheral nerve sheath tumor (MPNST) cells Appropriate number of cells that can reach -70% confluence in 3 days (2000-6000 cells/well) were plated in 96-well
  • 53412 v2OC 149 cells were then cultured for 3 days.
  • a positive control compound (17-AAG) and a vehicle (DMSO) control were included with each assay.
  • DMSO a vehicle
  • ATPlite solution Perkin Elmer
  • Viable cells were measured by detecting luminescence generated from reaction of ATPlite solution and the ATP in the cells.
  • IC50 the concentration needed for 50% inhibition of cell proliferation, were determined and analyzed for each compound using XLfit 4.1 software.
  • Efficacious compounds preferably have about an IC50 ⁇ lO ⁇ M. IC50s of these compounds inhibiting proliferation of various NFl and NF2 cells are shown in Figure 3.
  • the present invention also includes screening for degradation of one or more client proteins of HSP90 or upregulation of HSP70 using peripheral blood mononuclear cells ("PMBC PD assay " ).
  • PMBC PD assay whole blood from groups of about 5 similarly treated normal and/or mice harboring NF2-deficient or NFl -deficient tumors at efficacious doses is collected and pooled in a single heparinized Vacutainer tube (BD biosciences, NJ), and peripheral blood mononuclear cells (PBMCs) are isolated as reported (Graff et ai, 2005, Cancer Res. 65(16): 7462-7469; Peralba et al, 2003, Clin. Cancer Res. 9(8): 2887-2892).
  • PBMCs are collected at 0, 2, 4, 8, and 24 hours post dosing. PBMCs are lysed directly for Western blots or for immunoprecipitation first and then Western blots to detect target proteins. For example, HSP70 upregulation by a HSP90 inhibitor is detected in PBMCs (Ramanathan et al., 2007, Clin. Cancer Res. 13(6): 1769-1774).
  • HSP70 upregulation by a HSP90 inhibitor is detected in PBMCs (Ramanathan et al., 2007, Clin. Cancer Res. 13(6): 1769-1774).
  • target protein degradation e.g. ErbB2, c-Raf, Akt, Cdk4
  • phosphorylation phosphorylation
  • HSP70 upregulation in tumor lysates by Western blots or in tumor sections (i.e.
  • paraffin-embedded sections by immunohistochemistry are used to assay the effects of the test compound on target proteins.
  • Tumor tissues are harvested at the same time points as described above for PBMCs. Each tumor sample is divided in two pieces with one flash frozen in liquid nitrogen immediately and the other fixed in 10% buffered formalin and then paraffin-embedded.
  • ErbB2, phospho- Akt, and phospho-S6 levels in Np -/-Schwann cell xenografts have been shown to be veiy high and can be readily detected by immunohistochemistry (reference is made to a PCT application entitled 'Treatment of Neurofibromatosis with Inhibitors of a Signal Transduction Pathway" referenced as Attorney Docket No. NEXG-005-01 WO, filed on June 4, 2007).
  • test compound to act as a therapeutic in NF2 or NFl by modulating the HSP90 complex can also be assessed using animal xenograft experiments.
  • Xenograft experiments are performed with subcutaneous tumor formation in nude mice or SCID mice using a human NF2-deficient malignant mesothelioma cell line RAV, a meningioma cell line or Nf2-/- SCs.
  • a dermal neurofibroma (DNF) xenograft model has been established to evaluate compounds useful for treating NFl neurofibromas (PCT publication WO 06/083979). In particular, these experiments have allowed the inventors of the present invention to define the treatment starting point for each xenograft model.
  • tumor-bearing mice are randomized among control and treated groups with 8- 12 mice in each experimental group.
  • compounds are suspended in 0.5% (w/v) methyl cellulose or other suitable dosing vehicles known in the art. Dosing starts from a predefined starting point for each cell system (i.e. 100-200 mm 3 at 3-5 days for Nf2-/- Schwann cells). Mice are dosed once or twice a day with high, medium, low doses and vehicle control for 4 weeks or less determined by preset tumor volume criteria.
  • mice are dosed twice daily, once daily, three times a week, two times a week, once a week. In case of significant variations in body weight during the treatment period, doses can be adjusted accordingly.
  • mice are randomized among control and treated groups with 15-20 mice in each experimental group. Mice are dosed every week day with high, medium, and low doses and vehicle control for 8 weeks or less beginning at 18-20 weeks of age, when 30-50% of mice begin to develop tumors. The high dose is defined as the MTD dose of the test compound with similar schedule.
  • test agents are administered via intraperitoneal (i.p.), intravenous (i.v.), or oral route. In case of significant variations in body weight during the treatment period, doses are adjusted accordingly or experiments can be repeated with the adjusted doses.
  • cisNfl ;p53 mice that develop tumors become less active and assume classic rounded positions and ruffled coat. An untreated cohort of mice is expected to begin to develop signs of ill health within the fifth month of age.
  • Pairs of control and test mice are sacrificed on a weekly basis once the signs of illness become apparent in control mice. All mice are carefully necropsied and the number, location and size of all tumors are noted. All tumor samples are evaluated for histologic and immunohistochemical analysis to determine pathology as well as evidence for apoptosis, necrosis, and proliferation.
  • Hepta-2,6-dienoic acid methoxy-methyl-amide (2-1 14): To a solution of 2-chloro-yV-methoxy- vV-methylacetamide (6.0 g, 48.8 mmol) in dry DMF (20 mL) at 23 0 C was added 3- mercaptophenol (4.44 mL, 48.8 mmol) and K 2 CO 3 (6.7 g, 48.8 mmol). The resulting suspension was stirred at 23 0 C overnight.
  • the final mass gain (5.2 g, 27.3 mmol) indicated an estimate loading of 0.81 mmol.g "1 .
  • Resin 2-49 (10 g, 0.81 mmol.g “1 ) was suspended in a 1 : 1 mixture Of HFIPZCH 2 Cl 2 (50 mL). To this suspension, H 2 O 2 (3 mL, 16.0 mmol) was added at 23 0 C and the resulting mixture was shaken for 12 h. Resin 2-113 was then filtered, washed using MeOH (50 mL), CH 2 Cl 2 (50 mL) and Et 2 O (50 mL) and dried under reduced pressure to constant mass before subsequent use.
  • Resin 2-113 (4.0 g, ⁇ 0.81 mmol.g " 1 ) was suspended in DMSO (40 mL) followed by the addition of fBuOK (336 mg, 3.0 mmol). After shaking the reaction for 1 h at room temperature, 5-iodo-l-pentene (588 mg, 3.0 mmol) was added to the suspension and the mixture was shaken for 3 h. The resin was filtered, washed and dried as before. Then, it was suspended in toluene and heated at 80 0 C. After 8 h at this temperature,
  • Macrocycle 2-129 An aqueous Na 2 ⁇ EDTA solution (700 ⁇ L, 4 x 10 "4 M) was added to a solution of compound 2-128 (80 mg, 0.14 mmol) in a 2: 1 mixture of dimethoxymethane/acetonitrile (2.1 mL). The resulting mixture was cooled to 0 0 C and treated with trifluoroacetone (150 ⁇ L) added via a precooled syringe. A mixture of sodium bicarbonate (88 mg, 1.05 mmol) and Oxone (430 mg, 0.70 mmol) was added in portions over a period of ⁇ 1 h to this homogeneous solution. The reaction was followed by TLC and found to be complete in 2 h.
  • Macrocycle 2-132 A 2 niM solution of compound 2-131 (166 mg, 0.26 mmol) in anhydrous toluene (130 mL) was treated with 10 % mol of Grubbs' II (20.8 mg, 0.026 mmol) and heated up to 80 0 C for 12 h. The reaction mixture was then filtered through a pad of silica, which was washed with CH 2 Cl 2 . The combined filtrates were concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-25 % EtOAc/hexane gradient) afforded macrocycle 2-132 (136 mg, 87 %).
  • Macrocycle 2-133 An aqueous Na 2 *EDTA solution (350 ⁇ L, 4 x 10 "4 M) was added to a solution of compound 2-132 (40 mg, 65 ⁇ mol) in a 2: 1 mixture of dimethoxymethane/acetonitrile (1.1 mL). The resulting solution was cooled to 0 0 C and trifluoroacetone (75 ⁇ L) was added via a precooled syringe. A mixture of sodium bicarbonate (44 mg, 0.5 mmol) and Oxone (215 mg, 0.35 mmol) was then added in portions over a period of ⁇ 1 h to this homogeneous solution. The reaction was followed by TLC and found to be complete in 2 h.

Abstract

The present invention includes methods of treating NF2-deficient or NF1-deficient cells or neurodegenerative diseases with radicicol or its derivatives, such as one or more compounds of formula I, II, III, IV, or V. The present invention is also directed to methods of inhibiting the growth of NF2-deficient or NF1-deficient tumors. The methods comprise contacting NF2-deficient or NF1-deficient tumor cells with radicicol or its derivatives, such as one or more compounds of formula I, II, III, IV, or V. The present invention is also directed to the combinational use of radicicol or its derivatives, such as one or more compounds of formula I, II, III, IV, or V with at least one additional active agent, such as one or more HSP90 inhibitors.

Description

TREATMENT OF NEUROFIBROMATOSIS WITH RADICICOL AND ITS DERIVATIVES
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to International Application No.
PCT/US2007/70367, filed June 4, 2007, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION [0002] The present invention relates to the use of radicicol and its derivatives for treatment of neurofibromatosis type 2 (NF2), neurofibromatosis type 1 (NFl), and neurodegenerative diseases.
BACKGROUND OF THE INVENTION [0003] Neurofibromatosis includes two diseases, neurofibromatosis type 1 (NFl) and neurofibromatosis type 2 (NF2). Both NFl and NF2 are inherited disorders and both encompass mutations which predispose individuals to multiple tumors of the central or peripheral nervous system, and occasionally to other malignancies. Major tumor types associated with NFl and NF2 involve glial cells (e.g. Schwann cells and astrocytes). Although there is the similarity of the involvement of Schwann cells in NFl and NF2 tumors, NFl and NF2 have a spectrum of tumors which involve different types of cells. In addition, NFl and NF2 are caused by different gene mutations. Neurofibromatosis type 2 [0004] Neurofibromatosis type 2 (NF2) is a rare form of neurofibromatosis, which is a dominantly inherited tumor suppressor disorder, that affects approximately 1 in 25,000 individuals and is characterized by multiple tumors on the cranial and spinal nerves. NF2 is a different disease from NFl , neurofibromatosis type 1. Although both NFl and NF2 are tumor predisposition syndromes in the nervous system, the tumor suppressor genes are different and signaling pathways are likely to be different. [0005] Individuals with NF2 are at a high risk for developing brain tumors, in particular tumors on both the seventh and eighth cranial nerves. Bilateral vestibular schwannomas, a type of tumor which occurs on these nerves, occurs in about 95% of affected individuals. Consequently, hearing loss, ringing in the ears, and problems with balance are symptoms frequently associated with NF2.
53412 DC 1 [0006] Schwannomas are tumors consisting of nerve sheath cells or Schwann cells (SCs). Schwann cells support and protect nerve cells and provide nerves with the insulation they need to conduct infoπnation. Bilateral vestibular schwannomas, also known as acoustic neuromas, as well as spinal schwannomas and schwannomas of the peripheral nerves are common manifestations of NF2. The symptoms of a schwannoma will depend on its location.
[0007] In addition to schwannomas, individuals with NF2 may develop other types of tumors emanating from the nerves, meningeal envelopes, brain and spinal cord. The most common tumor of this type is meningioma; other less common tumors include ependymomas and astrocytomas. Moreover, NF2 patients may have an increased risk for developing mesotheliomas. Molecular Role of Merlin
[0008] NF2 is an autosomal dominant genetic trait, meaning it affects both genders equally and each child of an affected parent has a fifty percent chance of inheriting the gene. NF2 results from a mutation or a deletion of the NF2 gene and is transmitted on chromosome 22 (Sainz e/ fl/., 1994, Hum. MoI. Genet. 3: 885-891 ; Ruttledge et al., 1994, Nat. Genet. 6: 180- 184; Rubio et al., 1994, Cancer Res. 54: 45-47; Huynh et al, 1997, J. Neuropathol. Exp. Neurol. 56: 382-390), [0009] The NF2 gene is a tumor suppressor gene that encodes a 595-amino acid protein, termed Merlin. Merlin belongs to the ezrin, radixin, and moesin (ERM) family of proteins (Trofatter et al., 1993, Cell. 75: 826).
[0010] Over-expression of Merlin can block both cell proliferation and oncogene-induced transformation (Lutchman and Rouleau, 1995, Cancer Res. 55(1 1): 2270-2274; Tikoo et al., 1994, J. Biol. Chem. 269(38): 23387-23390). Indeed, Merlin can negatively regulate cyclin D 1 levels (Xiao et al., 2002, J. Biol. Chem. 277: 883-886) and loss of Merlin results in overexpression of cyclin Dl (Lallemand et al., 2003, Genes Dev. 17: 1090-1 100). However, given its predominant localization to the membrane and cytoskeleton interface, Merlin is not likely to directly control the cell cycle machinery. [0011] The mechanism by which loss of NF2 may contribute to tumor development is not clear. Many studies have focused on this issue using both genetic and biochemical approaches. Several lines of evidence suggest that Merlin can regulate receptor tyrosine kinase activity, trafficking, and degradation. Merlin has been shown to interact directly with the focal adhesion component paxillin in a complex that contains integrin-βl and ErbB2 (Fernandez- Valle et al, 2002, Nat. Genet. 31(4): 354-362), HGF receptor substrate (HRS)
53412 v2, DC 2 (Scoles et al, 2002, Hum. MoI. Genet. 1 1(25): 3179-3189; Gutmann et al., 2001 , Hum. MoI. Genet. 10(8): 825-834; Soles et al, 2000, Hum. MoI. Genet. 9(1 1): 1567-1574), and platelet derived growth factor receptor (PDGFR) indirectly through interaction with a PDZ-containing adaptor protein EBP50/NHE-RF (Maudsley et al, 2000, MoI. Cell Biol. 20(22): 8352-8363; Mmthy et al, 1998, J. Biol. Chem. 273(3): 1273-1276). Neuregulin growth factors (EGF family of growth factors), VEGF, and HGF are important mitogens for Schwann cells (SCs) (Krasnoselsky et al, 1994, J. Neurosci. 14:7284-7290; DeClue et al, 2000, J. Clin. Invest. 105(9): 1233-1241 ; Caye-Thomasen et al, 2005, Otol. Neurotol. 26(l):98-101 ). Neuregulin/ErbB pathways are constitutively activated in human NF2 vestibular schwannomas and inhibitors of these pathways (e.g. antibody against neuregulin and Iressa) block proliferation of NF2-deficient schwannoma cells (Stonecypher et al, 2006, J. Neuropathol. Exp. Neurol. 65:162-175; Hansen et al., 2006, Glia 53:593-600). Recent evidence from Drosophila indicates that Merlin can regulate abundance/turnover of many signaling and adhesion receptors such as Notch, the EGF receptor, Patched, Smoothened, E- cadherin, and Fat. Loss of merlin results in accumulation of these cell surface receptors and activation of the associated signaling pathways (e.g. the EGFR pathway and the Wingless pathway) (Maitra et al, 2006, Curr, Biol. 16(7):702-709).
[0012] In addition to cell surface receptors, Merlin has been shown to interact with downstream components of various signaling pathways, including Rac-PAK (p21 -activated kinase) pathway. Rac is a member of the Rho family of small GTPases, which organize the actin cytoskeleton and control many cellular processes such as cell proliferation, transformation, and cell motility (Etienne-Manneville and Hall, 2002, Nature. 420(6916): 629-635; Sahai and Marshall, 2002, Nat. Rev. Cancer 2(2): 133-142). PAK can phosphorylate S518 of Merlin (Xiao et al, 2002, J. Biol. Chem. 277: 883-886; Kissel et al, 2002, J. Biol. Chem. 277(12): 10394-10399) which leads to conformational change and loss of growth-suppressing activity (Shaw et al, 1998, J. Biol. Chem. 273(13): 7757-7764; Shaw et al, 2001 , Dev. Cell. 1 (1): 63-72). Merlin can also act as a negative regulator of Rac-PAK signaling (Shaw et al, 2001 , Dev. Cell. 1 : 63-72; Kissil et al, 2003, MoI. Cell. 12:841-849; Lallemand et al., 2003, Genes Dev. 17: 1090-1 100; Hirokawa et al., 2004, Cancer J. 10: 20- 26). Loss of Merlin results in the inappropriate phosphorylation and activation of PAK.
Over-expression of Merlin inhibits PAK activation and blocks Rac-induced transformation. (Shaw et al, 2001 , Dev. Cell. 1 (1 ): 63-72; Kissil et al, 2003, MoI. Cell. 12(4):841 -849). Preliminary evidence indicates that loss of Merlin also leads to activation of the Ras/Raf/Mek/Erk pathway and PI3K-Akt pathway (Rangwala et al, 2005, J. Biol. Chem.
53412 v2 DC T 280( 12): 1 1790-1 1797; our preliminary data). A recent study from Drosophila has proposed that Merlin and a related protein expanded function upstream of the Hippo signaling pathway to regulate cell proliferation and apoptosis (Hamaratoglu et a!., 2006, Nat. cell biol. 8:27-36; Willecke et a!., 2006, Curr Biol. 16(21):2090-2100). [0013] The link between Merlin and growth factor receptor signaling indicates that growth factor receptors may play direct roles in NF2-associated tumor formation and progression. However, possible involvement of Merlin with multiple signaling pathways presents a challenge in developing drugs for the treatment of NF2. Neurofibromatosis type 1 [0014] NFl is one of the most common single gene disorder to affect the human nervous system, with an incidence of 1 in 3500 individuals (Sorensen SA, Mulvihill JJ, Nielsen A. Ann N Y Acad Sci 1986;486:30-7.). NFl affects approximately 1.5 million people worldwide and there is no racial, ethnic, or geographic predilection for the disease. NFl is an autosomal dominantly inherited genetic disorder with frequent gemiline deletion or loss-of- function mutations of the NFl gene, and is caused by mutation in the NFl gene, which encodes Neurofibromin, a tumor suppressor. Neurofibromin shares a region of similarity with the pl20RasGAP protein, therefore functioning as a negative regulator of the Ras pathway. A high spontaneous mutation rate (50%) at the NFl locus and the substantial variability of its expression ensure that the disorder is unlikely to decrease significantly in the population due to genetic screening.
[0015] The signs of NFl include cafe-au-lait macules, skin freckling, skeletal defects, learning disability, Lisch nodules, dermal and plexiform neurofibromas (most common), benign tumors of the brain or other organs (e.g. optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ganglioneuromas, ependymomas, pheochromocytomas and ganglioneuromas), and malignant neoplasms (e.g. rhabdomyosarcomas, neurofibrosarcomas or malignant peripheral nerve sheath tumors ("MPNST") or malignant schwannomas) (Korf BR. J Child Neurol 2002; 17(8):573-7; discussion 602-4, 46-51.) Children affected by NFl also have increased risk for developing a rare form of leukemia-juvenile myelomonocytic leukemia (JMML) (Stiller CA, Chessells JM, Fitchett M. Br J Cancer 1994;70(5):969-72.). Dermal neurofibromas, subdermal neurofibromas, plexiform neurofibromas and MPNSTs are primarily derived from Schwann cells or their progenitors. Optic gliomas and astrocytomas are derived from astrocytes. Pheochromocytomas are derived from neural crest components (as are neurofibromas and MPNSTs).
53412 v2 DC A [0016] The typical characteristic of NFl is the neurofibroma, of which there are clinically and histologically distinct types. Ninety- five% of patients have discrete benign neurofibromas within the dermis which may develop at any time in life, but their numbers are usually small before puberty. The total number of neurofibromas seen in adults varies from just a few to hundreds or even thousands. These tumors may cause disfigurement, chronic pain and pruritus. Certain patients may develop some of the same disfiguring symptoms that are associated with Elephant Man's disease, a separate disorder originally thought to be NFl . Plexiform neurofibromas may be congenital and are present in 30% of patients with NFl . These tumors represent a major cause of morbidity in NFl . They affect long portions of nerves and infiltrate the nerve and surrounding tissue, resulting in disfiguration and neuralgic complications. In about 2-5% of patients, plexiform neurofibromas transform to malignant peripheral nerve-sheath tumors, which have a significant mortality rate. Although NFl is usually not a lethal disorder, affected individuals often face a lifetime of morbidity and disfigurement. [0017] The NfI gene was identified in 1990 (Wallace et al. 1990 Science 249: 181-186; Cawthon et al. 1990 Cell 62: 193-201) and its gene product, neurofibromin, is a 250 kD protein of 2818 amino acids that has a catalytic domain related to the GTPase-activating protein (GAP) domain of p!20RasGAP (Marchuk et al., 1991 Genomics 1 1 :931-940; Gutmann et al., 1991. Proc. Natl. Acad. Sci. U. S. A. 88: 9658-9662; DeClue et al., 1991. Proc. Natl. Acad. Sci. U. S. A. 88:9914-9918; Martin et al., 1990. Cell 63:843-849; Xu et al., 1990. Cell 63: 835-841 ; Xu et al., 1990. Cell 62: 599-608). Loss of NfI in human neurofibromas, MPNSTs, leukemias, and tumor-derived cell lines results in the elevation of Ras-GTP levels and activation of Ras-Raf-Mek-Erk2 and other MAP kinase pathways (Guha et al., 1996 Oncogene 12: 507-513; Bollag et al., 1996. Nat. Genet. 12: 144-148; Basu et al. 1992. Nature 356: 713-715; DeClue et al., 1992 Cell 69:265-273). For example, Ras-GTP levels from a few NFl MPNST-derived cell lines ST88-14, 88-3 and 90-8 are much higher compared to other cell lines with normal neurofibromin. These cell lines also have activated downstream MAP kinase pathways. In addition, cell proliferation and soft agar growth of ST88-14 can be inhibited by injection of an antibody against Ras and expression of the GAP domain of neurofibromin, respectively. Therefore, controlling Ras pathway activity in these cells is important in blocking the transformation properties. HSP90 andHSP90 Inhibitors/Modulators
[0018] The heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone whose function is to ensure the proper folding and stability of a number of its client proteins
53412 v2/ DC S such as kinases and transcription factors (Pearl and Prodromou, 2001 Adv. Protein Chem, 59: 157-186). HSP90 belongs to the ATPase superfamily and consists of three protein domains: the N-terminal ATPase domain, a middle domain responsible for client protein binding, and a C-terminal dimerization domain which also contains a weak ATP-binding domain (Pearl and Prodromou, 2001 Adv Protein Chem. 59: 157-186). Four genes of the HSP90 family are found in humans and their gene products have different cellular locations. The two major cytoplasmic i so forms are HSP90 alpha and HSP90 beta (Hickey et al., 1989, MoI Cell Biol. 9: 2615-2626). Other major isoforms are GRP94 in the endoplasmic reticulum (Argon and Simen, 1999, Semin, Cell Dev. Biol. 10: 495-505) and TRAP1/HSP75 in mitochondria (Felts et al., 2000, J. Biol. Chem. 275: 3305-12). HSP 90 is found to be part of a series of dynamic multiprotein complexes made of co-chaperones including HSP70, HSP40, and Hop. Hydrolysis of ATP causes HSP90 to alter its conformation and allows other co- chaperones such as p23, CDC37, or imunophilins to associate with HSP90 to form a mature complex, which catalyzes the folding and maturation of the client proteins (Pearl & Prodromou, 2000, Curr. Opin. Struct. Biol. 10: 46-51.). The adaptor co-chaperone protein CDC37 mediates interactions between HSP90 and kinases (Pearl, 2005, Curr. Opin. Genet. Dev. 15:55-61 ; Roe et al., 2004, Cell 1 16: 87-98.).
[0019] There are over one hundred HSP90 client proteins reported in the literature (Solit and Rosen, 2006, Curr. Top. Med. Chem. 6: 1205-14). Major HSP90 client proteins include steroid hormone receptors such as the androgen, estrogen and glucocorticoid receptors (AR, ER, and GR) (Whitesell and Cook, 1996, MoI. Endocrinol. 10: 705-712; Segnitz and Gehring, 1997, J Biol Chem. 272(30): 18694-701 ; Czar et al., 1997 Biochemistry. 1997, 36:7776-85), tyrosine and serine/threonine kinases such as HER2 (ErbB2) (Munster et al., 2002, Cancer Res. 62: 3132-3137.), the insulin-like growth factor- 1 receptor (IGF-IR) (Sepp- Lorenzino et al., 1995, J. Biol. Chem. 270: 16580-16587.), Met (Webb et al., 2000, Cancer Res. 60: 342-349.), Flt-3 (Yao et al., 2003, Clin. Cancer Res. 9: 4483-4493.), ZAP70 (Castro et al., 2005, Blood 106: 2506-2512.), Src family kinases (Bijlmakers and Marsh, 2000, MoI Biol Cell. 1 1 : 1585-1595.) Raf-1 (Schulte et al., 1995, J. Biol. Chem. 270: 24585-24588.), cyclin-depcndent kinases 4 and 6 (Cdk4/6) (Stepanova et al., 1996, Genes Dev. 10: 1491 - 1502.), MLK3 (Zhang et al., 2004, J. Biol. Chem. 279: 19457-19463.), and Akt (Basso et al., 2002, J. Biol. Chem. 277: 39858-39866.), mutant proteins including v-Src (Xu et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7074-7078; Whitesell et al., 1994, Proc. Natl. Acad. Sci. USA 91 :8324-8328), mutant EGFR (Shimamura et al., 2005, Cancer Res. 65: 6401 -6408.), mutant B-Raf (da Rocha Dias et al., 2005, Cancer Res. 65: 10686-10691 ; Grbovic et al., 2006, Proc.
53412 \2'DO f, Natl. Acad. Sci. USA 103: 57-62.), Bcr-Abl (Gorre et al., 2002, Blood 100: 3041 -3044; Nimmanapalli et al., 2001 , Cancer Res. 61 : 1799-1804.) and mutant p53 (Whitesell et al., 1998, MoI. Cell Biol. 18: 1517-1524.), and other proteins such as HIF-I alpha (Isaacs et al., 2002, J. Biol. Chem. 277: 29936-29944; Mabjeesh et al., 2002, Cancer Res. 62: 2478-2482.), Mdm2 (Peng et al., J. Biol. Chem. 276: 40583-40590.), and HSF-I (Zou et al., 1998, Cell 94: 471 -480.). Many of these HSP90 client proteins are important in controlling cell growth and proliferation, differentiation, and cell survival. This is a putative rationale for the use of HSP90 inhibitors in the treatment of cancer. This subject has been extensively reviewed in the recent literature (Chiosis and Neckers, 2006, ACS. Chem. Biol. l(5):279-284; Janin, 2005, J. Med. Chem. 48(241:7503-7512; Sharp and Workman, 2006, Adv. Cancer Res. 95:323-348; Solit and Rosen, 2006, Curr. Top. Med. Chem. 6(11): 1205-1214). The identification of specific HSP90 clients that are important for the growth of specific cancer types is an active area of research. [0020] Two structurally unrelated natural products, geldanamycin and radicicol, were isolated in 1970 from Streptomyces hygrocopicus and in 1953 from the fungus Monospoήum bonorden, respectively. Geldanamycin is a benzoquinone-based ansamycin antibiotic, and radicicol is a macrocyclic lactone antibiotic. They were believed to be kinase inhibitors initially and later found to inhibit HSP90 function via the interaction with the N-terminal ATP binding domain of HSP90 (DeBoer et al., 1970, J. Antibiot. 23: 442-447, Roe et al ., 1999, J. Med. Chem. 42:260-266; Schulte and Neckers, 1998, Cancer Chemother. Pharmacol,
42:273-279; Prodromou et al., 1997, Cell 90:65-75; Whitesell et al., 1994, Proc. Natl. Acad. Sci. U. S. A. 91 :8324-8328). More compounds have been found to inhibit the function of HSP90 and are generally referred to as HSP90 inhibitors. Most HSP90 inhibitors inhibit the intrinsic ATPase activity by binding to the N-terminal nucleotide binding site of HSP90 and thus block the formation of the mature complex between HSP90, co-chaperones, and the client proteins since the formation of the mature complex is dependent on ATP hydrolysis. The client proteins are then degraded through the ubiquitin-proteasome degradation pathway (Connell et al., 2001 , Nat. Cell Biol. 3: 93-96.). The coumarin antibiotic novobiocin binds to the C-terminal ATP-binding site of HSP90 but with a very weak activity to degrade HSP90 client proteins (Marcu et al., 2000, J. Natl. Cancer Inst. 92:242-248; Marcu et al., 2000, J. Biol. Chem. 275:37181-37186).
[0021] Both geldanamycin and radicicol demonstrate good cellular potency but are not suitable for clinical development. Geldanamycin has severe hepatotoxicity and radicicol is not stable in serum thereby having no in vivo anti-tumor activity (Agatsuma et al., 2002, Bioorg.
53412 v2 DC 7 Med. Chem. 10:3445-3454; Soga et al., 2003, Curr. Cancer Drug Targets, 3:359-369). Thus, analogs of radicicol, such as oxime derivatives of radicicol, and analogs of geldanamycin, such as 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), the more water soluble analogue 17-demethoxy, 17-(2-dimethylamino) ethylamino geldanamycin (17-DMAG), and hydroquinone analogue of 17-AAG, have been synthesized and tested. These analogs generally exhibit good efficacy at tolerated doses in in vitro studies and in vivo animal xenograft models (Ikuina et al., 2003, J. Med. Chem., 46:2534-2541 ; Ge et al., 2006, J. Med. Chem. 49:4606-4615; Maroney et al.. 2006. Biochemistry. 45:5678-5685; Shiotsu et al., 2000, Blood, 96(6):2284-2291 ; Sydor et al., 2006. Proc. Natl. Acad. Sci. U.S.A. 103(46): 17408-17413). Several geldanamycin analogs have entered clinical trials for the treatment of cancer. These agents have demonstrated promising results in several types of cancer (e.g. breast cancer, leukemia, melanoma, and etc.).
[0022] The clinical experience of 17-AAG has stimulated a search for new HSP90 inhibitors with better pharmacological properties and safety profiles. A number of HSP90 inhibitors in various compound classes have been developed as potential agents for cancer treatment. These include purine-based compounds (PCT publications WO/2006/084030; WO/2002/036075; US7,138,401 ; US20050049263; Biamonte et al., 2006, J. Med. Chem. 49:817-828; Chiosis, 2006, Curr. Top. Med. Chem. 6: 1183-1191 ; He et al., 2006, J. Med. Chem. 49:381-390), pyrazole-based compounds (Rowlands et al., 2004, Anal. Biochem. 327: 176-183; Dymock et al., 2005, J. Med. Chem. 48:4212-4215; PCT publication WO/2007/021966; WO/2006/039977; WO/2004/096212; WO/2004/056782; WO/2004/050087; WO/2003/055860; US7, 148,228), peptidomimetic shepherdin (Plescia et al., 2005, Cancer Cell 7:457-468: US publication 20060035837), and HSP90 inhibitors in other compound classes (PCT publications WO/2006/ 123165; WO/2006/109085; WO/2005/028434; US7,160,885; US7,138,402; US7,129,244; US20050256183;
US20060167070; US20060223797; WO2006091963). Furthermore, small molecules that modulate HSP functions have been reported. For instance, HDAC inhibitors such as Trichostatin A, SAHA, and FK228 are capable of inhibiting deacetylation of HSP90 and thus modulating the function of HSP90 (Kovacs et al., 2005, Molecular Cell, 18: 601-607). Other molecules modulating the level of HSPs, such as HSP70 and HSP27 may also affect the function of the HSP90 complex (Zaarur et al., 2006, Cancer Res. 66(3): 1783-1791). None of these HSP90 inhibitors have previously been shown to inhibit the growth of NFl- or NF2- deficient tumor cells.
53412 v2 DC [0023] Current treatments for NF2- and NFl -associated tumors consist of surgical removal and focused-beam radiation. Neither treatment is considered optimal. Most patients with NF2 and NFl require multiple surgical and/or focused beam radiation procedures during their lifetime. Since the tumors of NF2 most frequently lie on nerves near the brain and spinal cord, their surgical removal is not without risk. For instance, surgical removal of vestibular schwannomas typically results in complete hearing loss and frequent facial nerve damage. Focused-beam radiation also has a significant incidence of hearing loss and facial nerve damage. Accordingly, a strong need exists for safer treatment options for NF2 and associated tumors (e.g. schwannomas, meningiomas, and mesotheliomas) and NFl and associated tumors (e.g. dermal neurofibromas, subdermal neurofibromas, plexiform neurofibromas, MPNSTs, gliomas, astrocytomas, and JMML). The present invention provides a novel method for treating NF2 and related NF2-deficient tumors and NFl and related NFl -deficient tumors and their associated signs and symptoms by administering radicicol or its derivatives.
SUMMARY OF THE INVENTION
[0024] In one embodiment, the present invention provides a method of treating, preventing or ameliorating tumors or symptoms resulting from neurofibromatosis in a subject suffering neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or neurofibromatosis type 1 (NFl) or a condition associated with the loss of NFl function comprising administering to said subject a therapeutically effective amount of at least one compound of formula I, II, III, IV or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof. [0025] In one embodiment, the present invention provides use of a compound of formula I, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof, for the manufacture of a medicament for the treatment, prevention, or amelioration of tumors or symptoms resulting from neurofibromatosis in a subject suffering neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or neurofibromatosis type 1 (NFl ) or a condition associated with the loss of NFl function. [0026] In one embodiment, the present invention provides a method of treating, preventing or ameliorating a neurodegenerative disease in a patient comprising administering to said patient a therapeutically effective amount of at least one compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof.
53412 v2/ DC [0027] In one embodiment, the present invention provides use of a compound of formula I,
II, III, IV, or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof, for the manufacture of a medicament for the treatment, prevention, or amelioration of a neurodegenerative disease,
[0028] In another embodiment, the present invention provides a method of inhibiting or reducing the growth or number of NF2-deficient tumor cells or NFl -deficient tumor cells comprising contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with at least one compound of formula I, II, III, IV or V, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof.
[0029] The compound of formula I has the following structure:
Figure imgf000011_0001
wherein:
R1 . R2, RJ, R4 and R""1 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkyl aryl, aralkyl, aryl, hetero alkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH2, NR2, SR, S(O)R, S(O)2R, -S(O)2NR2, -N(R)SO2R, -NC(O)R, -
NC(O)NR2, -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR2, -OC(O)OR, or -
OC(O)NR2;
A1 and A2 together are -CH2-CH2-, -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, 1 ,2-cycloρroρadiyl, or 1,2-oxirane; B1 and B2 together are -CH2-CH2- or Bi and B2 together represent a covalent bond;
X1 is hydrogen, halogen, OH, OR, NH2, NR2, NH-OR, SR, S(O)R, S(O)2R, -N-O- (CH2)H-CO2-R; or X1 together with X2 or X3 represents a covalent bond;
X2 and X3 are both hydrogen, or one of X2 and X3 is hydrogen and the other together with X1 represents a covalent bond; X4 and X5 together are =0, =S, =N-0R, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nCONR2,
=N-NR2, =N-N-S(O)R or =N-N-S(O)2R; or one of X4 and X5 is hydrogen and the other is
53412 v2 'DC 10 OH, OR, OC(O)R, OC(O)OR or OC(O)NR2; or one of X4 and X5 together with X6 represents a covalent bond and the other of X4 and X5 is OH, OR, OC(O)R, OC(O)OR, or OC(O)NR2;
X6 is hydrogen or X6 together with one of X4 and X3 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl, heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is O, 1 , 2 or 3. [0030] The compound of formula II has the following structure:
Figure imgf000012_0001
wherein, R7 is =0, =S, =N-0R, =N-0-(CH2)n-C(0)0R, =N-O-(CH2)n-C(O)NR2, =N-
NR2, =N-N-S(O)R or =N-N-S(O)2R.
[0031] The compound of formula III has the following structure:
Figure imgf000012_0002
wherein R is hydrogen, alkyl, arylalkyl, acyl or a protecting group. [0032] The compound of formula IV has the following structure:
53412 v2'DC
Figure imgf000013_0001
wherein R6 is hydrogen, OR, or NR2. [0033] The compound of formula V has the following structure:
Figure imgf000013_0002
> 9 • wherein R' is (CH2)πC(O)OR, or -(CH2)nC(O)NR2; and n is 0, 1 , 2 or 3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure I A shows graphs of Western blots demonstrating NXD30001 downregulates HSP90 client proteins and upregulates HSP70 in NF2-deficient mouse and human cells, while Figure 1 B shows graphs of Western blots demonstrating that the downregulation of HSP90 client proteins by NXD30001 is time-dependent.
J0035] Figure 2 shows graphs of actin immunoblots demonstrating the downregulation of HSP90 client proteins in ST88-14 NFl -deficient MPNST cell line resulted by NXD30001. [0036] Figure 3 shows the IC50 of radicicol derivatives, namely NXD30001 , NXD30002, and NXD30017, and the IC50 of 17AAG on cell proliferation of NFl and NF2 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Tumors in NF2 and NFl patients are unique in that they are slow growing tumors. Both NF2 and NFl tumors have mutations or loss of heterozygosity in tumor suppressor genes although the tumor suppressor genes are different- NF 2 or NFI genes, respectively. The inventors of the present invention have discovered that inhibitors of HSP90 potently blocked the proliferation of NF2-deficient and NFl -deficient tumor cells and also delay the growth of NF2-deficient and NFl -deficient tumors in mice. Merlin regulates the abundance
53412 v2< DC 12 and turnover of multiple cell surface receptors and interacts with multiple pathways. Many of these proteins are client proteins of HSP90. For instance, ErbB2 and other receptor tyrosine kinases, AKT, and Raf are well-established client proteins of HSP90. In addition, aberrant activation of the PI3K/AKT pathway has been found in human schwannomas from NF2 patients (as compared to normal nerves), in human NF2-defϊcient tumor xenografts (e.g. meningiomas and mesotheliomas), and in mouse Nf2-deficient Schwann cell tumors (reference is made to a PCT/US2007/70366 entitled 'Treatment of Neurofibromatosis with Inhibitors of a Signal Transduction Pathway," filed on June 4, 2007, which is incorporated in its entirety by reference). Neurofibromin is a negative regulator of Ras. Raf, the direct downstream effector of Ras, is a well-known client protein of HSP90. Neurofibromin has also been shown to regulate AKT. Therefore, a compound that inhibits HSP90 will likely be able to reduce the amount of AKT and other HSP90 client proteins such as ErbB2, IGF-IR, and Raf in the NF2-deficient cells or NFl -deficient cells. Reduction of the amount or activity of these proteins may be useful to reduce or stabilize the proliferation of NF2-deficient cells or NFl -deficient cells or to cause apoptosis of NF2-deficient cells or NFl -deficient cells. A compound that inhibits the activity of HSP90 is a compound that directly binds to the HSP90 protein or modifies the HSP90 protein post-translationally or regulate the transcription of HSP proteins such as HSP70 and HSP27 (Zaarur et al., 2006, Cancer Res. 66(3): 1783-1791). For instance, HSP90 can be acetylated and inactivated by histone deacetylase (HDAC) inhibitors (Kovacs et al., 2005, MoI. Cell 18(5):601-607; Fuino et al., 2003, MoI. Cancer Ther. 2:971-984; Aoyagi and Archer, 2005, Trends Cell. Biol. 15(1 1):565-567). For this reason, HSP90 inhibitory compounds are determined to be useful for the treatment of NF2-deficient tumors and NFl -deficient tumors which may not respond well to traditional chemotherapy and other cancer therapies which target fast growing and heterogeneous cancer cells. NF2- Associated Tumors
[0038] Patients with neurofibromatosis type-2 (NF2) have NF2-deficient tumors. This genetic characteristic, i.e., inactivation of the NF2 gene, differentiates tumors found in NF2 patients from genetically heterogeneous tumors such as breast and colon cancer tumors. For instance, NF2 patients have NF2-deficient meningiomas whereas some non-NF2-deficient meningiomas may contain mutations in many different oncogenes or tumor suppressor genes. [0039] As used herein, "NF2-deficient tumors" refer to tumors which contain a nonfunctioning NF2 gene. A non-functioning NF2 gene can be the result of a one or more insertion or deletion mutations within the NF2 gene, for instance, missense or nonsense mutations, mutations in the promoter or enhancer or introns that lead to no/low expression of
53412 v2 'DC 1 3 the NF2 gene, or the deletion of the entire NF 2 gene. NF2-deficient tumors are found in mesotheliomas and in patients with NF2, and include schwannomas, meningiomas, and other tumors associated with the nervous system. NF2-deficient tumors are also found in all patients with sporadic schwannomas and in 50%-70% of patients with meningiomas. [0040] The presence of NF2-deficient bilateral vestibular schwannomas, i.e., Schwann cell tumors, is a hallmark of NF2. The methods of the present invention can be used to inhibit the growth and/or kill NF2-deficient schwannoma cells, including those associated with vestibular schwannomas, spinal cord and other peripheral nerve schwannomas and sporadic schwannomas. Merlin and signaling proteins and pathways
[0041] As used herein, "NF2-deficient tumors" refer to tumors which contain a nonfunctioning NF2 gene. Merlin interacts with or regulates, but is not limited to, proteins and pathways such as Paxillin/Integrin-β l/ErbB2, EGFR, Patched/Smoothened, HRS, CD44, E- Cadherin, Fat, EBP50/NHE-RF/PDGFR, Wingless, Notch, Rac-PAK, PBK-AKT, Ras-Raf- Mek-Erk2, Hippo pathways, and downstream proteins thereof. Figure 1 is a schematic of involvement of Merlin with multiple cell surface proteins and signaling pathways. Targeting multiple proteins or pathways may be necessary for treating NF2. NFl- Associated Tumors
[0042] Patients with neurofibromatosis type- 1 (NFl) have NFl -deficient tumors. This genetic characteristic, i.e., inactivation of the NFl gene, differentiates tumors found in NFl patients from genetically heterogeneous tumors such as breast and colon cancer tumors. For instance, tumors found in NFl patients all have NFl gene mutations whereas patients with other cancers have mutations in different genes or overexpression of different genes. [0043] As used herein, "NFl -deficient tumors" refer to tumors which contain a non- functioning NFl gene. A non-functioning NFl gene can be the result of one or more insertion or deletion mutations within the NFl gene, for instance, missense or nonsense mutations, mutations in the promoter or enhancer or introns that lead to no/low expression of the NFl gene, or the deletion of the entire NFl gene. NFl -deficient tumors are found in patients with NFl , and include dermal, subdermal, plexiform neurofibromas, and MPNST and other tumors associated with the nervous system. NFl -deficiency also predisposes individuals to a rare form of leukemia, JMML.
[0044] NFl diagnosis is confirmed by fulfilling NIH clinical criteria or by finding an NFl mutation with mutational analysis. The methods of the present invention can be used to
53412 v2/DC 14 inhibit the growth and/or kill NFl -deficient tumors, including dermal, subdermal, plexiform neurofibromas, MPNST, gliomas, astrocytomas, pheochromocytomas and JMML.
Radicicol and Derivatives Thereof
[0045] The present invention provides the use of radicicol or its derivatives based on the resorcylic acid lactones, preferably compounds of formula I, II, III, IV or V, as HSP90 inhibitors for the treatment, prevention, or amelioration of tumors or symptoms resulted from neurofibromatosis. Also provided are compositions comprising the compounds and processes for the preparation of the compounds.
[0046] In one embodiment of the present invention, the compound of foπnula I has the following structure:
Figure imgf000016_0001
wherein:
R1 . R2, R"', R4 and R5 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkyl aryl, aralkyl, aryl, heteroalkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH2, NR2, SR, S(O)R, S(O)2R, -S(O)2NR2, -N(R)SO2R, -NC(O)R, - NC(O)NR2, -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR2, -OC(O)OR, or - OC(O)NR2; or R1 , R2, R3, and R5 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkyl aryl, aralkyl, aryl, heteroalkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH2, NR2, SR, S(O)R, S(O)2R, -S(O)2NR2, -N(R)SO2R, - NC(O)R, -NC(O)NR2, -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR2, -OC(O)OR, or - OC(O)NR2, and R4 has the structure of foπnula Ia: p
\— O-P-L1-R1 a L2
R2a Ia;
L1 and L2 are each independently a covalent bond, -0-, or -NR3a-;
53412 v2 DC 15 Rl a and R2a are each independently H, alkyl, cyclylalkyl, heteroaryl, heterocyclyl, alkenyl, alkynyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, -alkylene-C(O)-O-R4a, - (alkylene)-C(O)-O-R4a, -alkylene-O-C(O)-O-R4a, or -(alkylene)-O-C(O)-O-R4a; and
R3a and R4a are each independently H, alkyl, cyclylalkyl, heterocyclyl, heteroaryl, alkenyl, alkynyl, arylalkyl, or heterocyclylalkyl;
A1 and A2 together are -CH2-CH2-, -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, 1 ,2-cyclopropadiyl, or 1 ,2-oxirane;
B1 and B2 together are -CH2-CH2- or Bi and B2 together represent a covalent bond;
X1 is hydrogen, halogen, OH, OR, NH2, NR2, NH-OR, SR, S(O)R, S(O)2R, -N-O- (CH2)H-CO2-R; or X1 together with X2 or X3 represents a covalent bond;
X2 and X3 are both hydrogen, or one of X2 and X3 is hydrogen and the other together with X1 represents a covalent bond;
X4 and X5 together are =0, =S, =N-0R, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nCONR2, =N-NR2, =N-N-S(0)R or =N-N-S(0)2R; or one of X4 and X5 is hydrogen and the other is OH, OR, OC(O)R, OC(O)OR or OC(O)NR2; or one of X4 and X5 together with X6 represents a covalent bond and the other of X4 and X5 is OH, OR, OC(O)R, OC(O)OR, or OC(O)NR2;
X6 is hydrogen or X6 together with one of X4 and X3 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl, heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is O, 1 , 2 or 3.
[0047] In one preferred embodiment, R4 has the structure of formula Ia, which may increase the water solubility and/or bioavailability of the compound of formula I. [0048] In another embodiment of the present invention, the compound of formula I has the structure of formula II:
Figure imgf000017_0001
53412 v2/ DC 16 wherein, R7 is =0, =S, =N-OR, =N-O-(CH2)n-C(O)OR, =N-O-(CH2)π-C(O)NR2, =N- NR2, =N-N-S(O)R or =N-N-S(O)2R.
[0049] In one embodiment of the compound of formula II, R1 is H, halogen or heterocyclyl.
[0050] In another embodiment of the compound of formula II, R5 is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or arylalkyl.
[0051] In another embodiment of the compound of formula H, A1 and A2 together are
-CH=CH-.
[0052] In another embodiment of the compound of formula II, A1 and A" together are
-CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)- or -CH(halogen)-CH(OH)-. [0053] In another embodiment of the compound of formula II, A1 and A2 together are 1 ,2- oxirane.
[0054] In another embodiment of the compound of formula II, R1 is H, Cl or heterocyclyl;
R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are -CH=CH- or -C(OH)-C(OH)-; X1 is hydrogen, halogen or NH-OR; and R7 is =0, =S, =N-OR, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N-S(O)R, =N-N-
S(O)2R. It is preferable that R7 is =0. It is also preferable that R7 is =N-OR, =N-
O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2.
[0055] In another embodiment of the compound of formula II, R1 is H, Cl or heterocyclyl;
R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are 1 ,2-oxirane; X1 is hydrogen, halogen or NH-OR; and R7 is =0, =S, =N-OR, =N-
O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N-S(O)R, =N-N-S(O)2R.
[0056] In another embodiment of the compound of formula II, R1 is H, Cl or heterocyclyl;
R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are -CH=CH- or -C(OH)-C(OH)-; X1 together with X2 represent a bond; and R7 is =0, =S, =N-OR, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N-S(O)R, =N-
N-S(O)2R. Preferably, R1 is H or Cl; R5 is hydrogen, methyl, propyl, isopropyl or phenyl; and
R7 is =N-OR, =N-O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2 with R1 is Cl and R5 is hydrogen; or R7 is =N-OR, =N-O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2, and n is i ; or R5 is hydrogen and R7 is =N-O-(CH2)nC(O)OR, or =N-O-(CH2)πC(O)NR2; or R5 is hydrogen and R7 is =N-OR more preferable.
[0057] In another embodiment of the compound of formula II, R1 is H, Cl or heterocyclyl;
R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are 1 ,2-oxirane; X1 together with X2 represent a bond; and R7 is =0, =S, =N-0R,
=N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N-S(O)R, ^N-N-S(O)2R. It is
53412 v2/DC \ η preferred that R7 is =0. It is also preferred that R7 is =N-OR, =N-O-(CH2)nC(O)OR, or =N-
0-(CHz)nC(O)NR2.
[0058] In another embodiment of the present invention, the compound of formula I has the structure of formula III:
Figure imgf000019_0001
wherein R8 is hydrogen, alkyl, arylalkyl, acyl or a protecting group.
[0059] In one embodiment of the compound of formula III, R is hydrogen or acyl; and Ri is
H, halogen or heterocyclyl.
[0060] In another embodiment of the compound of formula III, R' is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or arylalkyl.
[0061] In another embodiment of the compound of formula III, X1 together with X2 represent a covalent bond.
[0062] In another embodiment of the compound of formula HI, wherein X1 is hydrogen, halogen, NH-OR NH-O-(CH2)nC(O)OR, or NH-O-(CH2)nC(O)NR2. [0063] In another embodiment of the compound of formula III, A1 and A2 together are
-CH=CH-.
[0064] In another embodiment of the compound of formula III, A1 and A2 together are
-CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)- or -CH(halogen)-CH(OH)-.
[0065] In another embodiment of the compound of formula III, Ai and Ai together are 1 ,2- oxirane.
[0066] In another embodiment of the present invention, the compound of formula I has the structure of formula IV;
53412 v2/DC 18
Figure imgf000020_0001
wherein R6 is hydrogen, OR, or NR2.
[0067] In one embodiment of the compound of formula IV, R is hydrogen or acyl.
[0068] In another embodiment of the compound of formula IV, R1 is H, halogen or heterocyclyl.
[0069] In another embodiment of the compound of formula IV, R5 is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or aryl alkyl.
[0070] In another embodiment of the compound of formula IV, A1 and A2 together are
-CH=CH-,
[0071] In another embodiment of the compound of formula IV, A1 and A2 together are
-CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)- or -CH(halogen)-CH(OH)-.
[0072] In another embodiment of the compound of formula IV, A1 and A2 together are 1,2- oxirane.
[0073] In another embodiment of the present invention, the compound of formula I has the structure of formula V:
Figure imgf000020_0002
V wherein R9 is (CH2)nC(O)OR, or -(CH2)nC(O)NR2; and n is 0, 1 , 2 or 3. [0074] In one embodiment of the compound of formula V, R6 is -CH2C(O)N(Me)OMe. [0075] In another embodiment of the compound of formula V, R1 is H, halogen or heterocyclyl.
[0076] In another embodiment of the compound of formula V, R5 is hydrogen, alkyl, lower alkyl, aryl, heteroaryl or arylalkyl.
53412 V2/DC 19 [0077] In another embodiment of the compound of formula V, A and A~ together are
-CH=CH-.
[0078] In another embodiment of the compound of formula V, A1 and A2 together are
-CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)- or -CH(halogen)-CH(OH)-.
[0079] In another embodiment of the compound of formula V, A1 and A2 together are 1 ,2- oxirane.
[0080] It should be understood that each of the R group described above can be independently the same or different. For example, -NR2 can be -N(RX)2, -N(Ry)2, or -NRxRy.
[0081 ] In specific embodiments of the present invention, the compounds presented in Table
1 or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof are provided:
Figure imgf000021_0001
53412 v2/D0 20
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
53412 v2 DC 23
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
53412 v2/DC 26
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
53412 v2/DC 37
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Pharmaceutically Acceptable Salts and Prodrugs
[0082] Nonlimiting examples of pharmaceutically acceptable salts are (a) acid addition salts formed with inorganic acids such as sulfate, nitrate, bicarbonate, and carbonate salts (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids including tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α- glycerophosphate salts, such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalcturonic acid; (b) base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, lithium and the like, or with a cation formed from ammonia, N,N-dibenzylethylenediamine, D-glucosamine, tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and (b); e.g., a zinc tannate salt or the like. Also included in this definition are pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula -NR^A", wherein R is as defined above and A is a counterion, including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).
[0083] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. [0084] Pharmaceutically acceptable "prodrugs" refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, animated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, dephosphorylated to produce the active compound.
Stereoisomerism and Polymorphism
[0085] Compounds of the present invention having a chiral center may exist in and be isolated in optically active and racemic forms. The present invention encompasses any
53412 v2 DC 45 racemic, optically-active, diastereomeric, polymorphic, or stereoisomer^ form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. [0086] In one embodiment, the compounds are prepared in optically active form by asymmetric synthesis using the processes described herein or synthetic transformations known to those skilled in the art.
[0087] Examples of methods to obtain optically active materials are known in the art, and include at least the following.
[0088] i) physical separation of crystals— a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct;
[0089] ii) simultaneous crystallization— a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; [0090] iii) enzymatic resolutions~a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; [0091] iv) enzymatic asymmetric synthesis-a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enatiomerically pure or enriched synthetic precursor of the desired enantiomer; [0092] v) chemical asymmetric synthesis— a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry
(i.e., chirality) in the product, which may be achieved using chrial catalysts or chiral auxiliaries;
[0093] vi) diastereomer separations~a technique whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; [0094] vii) first- and second-order asymmetric transformations—a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer;
53412 v2 DC 45 [0095] viii) kinetic resolutions— this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; [0096] ix) enantiospecific synthesis from non-racemic precursors—a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; [0097] x) chiral liquid chromatography— a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase. The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; [0098] xi) chiral gas chromatography— a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;
[0099] xii) extraction with chiral solvents— a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; or [00100] xiii) transport across chiral membranes— a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through. Definitions
[00101] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
[00102] When trade names are used herein, applicants intend to independently include the tradename product and the active pharmaceutical ingredient(s) of the tradename product. [00103] As used herein, "the compound", "compound(s) of the invention", or "compounds of formula I, II, III, IV, or V" means one or more compounds of formula I, II, III, IV, or V, or a pharmaceutically acceptable tautomer, salt, solvate, prodrug, or physiologically functional derivative thereof. Similarly, with respect to isolatable intermediates, such as for example, the phrase "a compound of formula (number)" means a compound of that formula and
53412 V2/DC 47 pharmaceutically acceptable tautomers, salts, solvates and physiologically functional derivatives thereof.
[00104] "Ester thereof means any ester of a nicotinic desensitizer in which any of the - COOH functions of the molecule is replaced by a -COOR function, in which the R moiety of the ester is any carbon-containing group which forms a stable ester moiety, including but not limited to alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and substituted derivatives thereof. The term "ester thereof includes but is not limited to pharmaceutically acceptable esters thereof, [00105] "Salt thereof means any acid and/or base addition salt of a compound of the present invention; preferably a pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salt" means a salt of a compound of the present invention which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, generally water or oil-soluble or dispersible, and effective for their intended use. Where applicable and compatible with the chemical properties of the nicotinic desensitizer, the term includes pharmaceutically-acceptable acid addition salts and pharmaceutically-acceptable base addition salts. Lists of suitable salts are found in, e.g., S. M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19. [00106] "Solvate thereof means a nicotinic desensitizer formed by solvation (the combination of solvent molecules with molecules or ions of the solute), or an aggregate that consists of a solute ion or molecule (the nicotinic desensitizer) with one or more solvent molecules.
[00107] "Chiral" refers to molecules which have the property of non-superimposability of the mirror image partner, while the term '"achiral" refers to molecules which are superimposable on their mirror image partner.
[00108] "Tautomers" refer to isomers of nicotinic desensitizers in which the isomers change into one another with great ease so that they ordinarily exist together in equilibrium. [00109] "Stereoisomers" refer to nicotinic desensitizers which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space. [00110] "Diastereomer" refer to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
53412 v2 DC 48 [00111] "Enantiomers" refer to two stereoisomers of a nicotinic desensitizer which are non- superimposable mirror images of one another.
[00112] Stereochemical definitions and conventions used herein generally follow S. N. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity. [00113] In the context of the present invention, protecting groups include prodrug moieties and chemical protecting groups.
[00114] Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e. routes or methods to prepare the compounds of the invention. For the most part the decision as to which groups to protect, when to do so, and the nature of the chemical protecting group "PG" will be dependent upon the chemistry of the reaction to be protected against (e.g., acidic, basic, oxidative, reductive or other conditions) and the intended direction of the synthesis. The PG groups do not need to be, and generally are not, the same if the compound is substituted with multiple PG. In general, PG will be used to protect functional groups such as carboxyl, hydroxyl, thio, or amino groups and to thus prevent side reactions or to otherwise facilitate the synthetic efficiency. The order of deprotection to yield free, deprotected groups is dependent upon the intended direction of the synthesis and the reaction conditions to be encountered, and may occur in any order as determined by the artisan. [00115] Various functional groups of the compounds of the invention may be protected. For example, protecting groups for -OH groups (whether hydroxyl, carboxylic acid, phosphonic
53412 v2/DC 49 acid, or other functions) include "ether- or ester-forming groups". Ether- or ester-forming groups are capable of functioning as chemical protecting groups in the synthetic schemes set forth herein. However, some hydroxyl and thio protecting groups are neither ether- nor ester- forming groups, as will be understood by those skilled in the art, and are included with amides, discussed below.
[00116] A very large number of hydroxyl protecting groups and amide-forming groups and corresponding chemical cleavage reactions are described in Protective Groups in Organic Synthesis, Theodora W. Greene and Peter G. M. Wuts (John Wiley & Sons, Inc., New York, 1999, ISBN 0-471-16019-9) (-'Greene"). See also Kocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), which is incorporated by reference in its entirety herein. In particular Chapter 1 , Protecting Groups: An Overview, pages 1-20, Chapter 2, Hydroxyl Protecting Groups, pages 21-94, Chapter 3, Diol Protecting Groups, pages 95-1 17, Chapter 4, Carboxyl Protecting Groups, pages 1 18-154, Chapter 5, Carbonyl Protecting Groups, pages 155-184. For protecting groups for carboxylic acid, phosphonic acid, phosphonate, sulfonic acid and other protecting groups for acids see Greene as set forth below. Such groups include by way of example and not limitation, esters, amides, hydrazides, and the like.
[00117] Ester-forming groups include: (1) phosphonate ester-forming groups, such as phosphonamidate esters, phosphorothioate esters, phosphonate esters, and phosphon-bis- amidates; (2) carboxyl ester-forming groups, and (3) sulphur ester-forming groups, such as sulphonate, sulfate, and sulfinate.
[00118] Also falling within the scope of this invention are the in vivo metabolic products of the compounds described herein. Such products may result for example from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabeled (e.g., C14 or HJ) compound of the invention, administering it parenterally in a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the metabolite), The metabolite structures are determined in conventional
53412 v2 DC 50 fashion, e.g., by MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well-known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the compounds of the invention even if they possess no anti- infective activity of their own.
[00119] Whenever a term in the specification is identified as a range (i.e. Ci-4 alkyl), the range independently refers to each element of the range. As a non-limiting example, C]-4 alkyl means, independently, Ci, C2, C3 or C4 alkyl. Similarly, when one or more substituents are referred to as being "'independently selected from'" a group, this means that each substituent can be any element of that group, and any combination of these groups can be separated from the group. For example, if R1 and R2 can be independently selected from X, Y and Z, this separately includes the groups R1 is X and R2 is X; R1 is X and R2 is Y; R1 is X and R2 is Z; R1 is Y and R2 is X; R1 is Y and R2 is Y; R1 is Y and R2 is Z; R1 is Z and R2 is X; R1 is Z and R2 is Y; and R1 is Z and R2 is Z. [00120] The term "alkyl" as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, including but not limited to groups with C1 to C)0.
[00121] The term "lower alkyl" refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, including groups with Ci to C4, and if appropriate a cyclic alkyl group (for example cyclopropyl).
[00122] Illustrative examples of alkyl groups are methyl, ethyl, propyl, wopropyl, cyclopropyl, butyl, secbutyl, isobutyl, tertbutyl, cyclobutyl, 1-methylbutyl, 1 ,1- dimethylpropyl, pentyl, cyclopentyl, isopentyl, neopentyl, cyclopentyl, hexyl, isohexyl, and cyclohexyl. Unless otherwise specified, the alkyl group can be unsubstituted or substituted with one or more moieties selected from the group consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, thiol, imine, sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfmyl, sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acid halide, anhydride, oxime, hydrazine, carbamate, phosphonic acid, phosphate, phosphonate, or any other viable functional group that does not inhibit the pharmacological activity of this compound, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al, Protective Groups in Organic Synthesis, John Wiley and Sons, 3rd Edition, 1999.
53412 v2'DC 51 [00123] The term "'halo" or "halogen", as used herein, includes chloro, bromo, iodo, and fluoro.
[00124] The term " alkyl thio" refers to a straight or branched chain alkylsulfide of the number of carbons specified, such as for example, C1.4alkyltb.io, ethylthio, -S-alkyl, -S-alkenyl, -S- alkynyl, etc.
[00125] The terms "alkylamino'Or "arylamino" refer to an amino group that has one or two alkyl or aryl substituents, respectively. Unless otherwise specifically stated in this application, when alkyl is a suitable moiety, then it is a lower alkyl, whether substituted or unsubstituted. [00126] The term "alkylsulfonyl" means a straight or branched alkylsulfone of the number of carbon atoms specified, as for example, Ci-6 alkylsulfonyl or methylsulfonyl. [00127] The term "alkoxycarbonyl" refers to a straight or branched chain ester of a carboxylic acid derivative of the number of carbon atoms specified, such as for example, a methoxycarbonyl, MeOC(O)-. [00128] As used herein, the term "nitro" means -NO2; the term "'sulfhydryl" means -SH; and the term "sulfonyl" means -S(O)T.
[00129] The terms "alkenyl" and "alkynyl" refer to alkyl moieties, including both substituted and unsubstituted forms wherein at least one saturated C-C bond is replaced by a double or triple bond. Thus, C2-6 alkenyl may be vinyl, allyl, 1-propenyl, 2-propenyl, 1 -butenyl, 2- butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, or 5-hcxenyl. Similarly, C2-6 alkynyl may be ethynyl, 1 -propynyl, 2- propynyl, 1 -butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1 - hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl. [00130] The term "alkyl ene" includes a saturated, straight chain, divalent alkyl radical of the formula -(CH2)H-, wherein "n" may be any whole integer from 1 to 10.
[00131] "Alkyl", "alkoxy", "alkenyl", "alkynyl", etc., includes both straight chain and branched groups. However, reference to an individual radical such as "propyl" embraces only that straight-chain radical, whereas a branched chain isomer such as "isopropyl" is specifically termed such. [00132] The term "aryl" as used herein and unless otherwise specified refers to any stable monocyclic, bicyclic, or tricyclic carbon ring of up to 8 members in each ring, wherein at least one ring is aromatic as defined by the Huckel 4n+2 rule, and especially phenyl, biphenyl, or naphthyl. The term includes both substituted and unsubstituted moieties. The aryl group can be substituted with any described moiety, including but not limited to one or more moieties
53412 v2/DC 52 selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, azido, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either protected or unprotected as necessary, as known to those skilled in the art, for example, as taught in Greene et al., Protective Groups in Organic Synthesis. John Wiley & Sons, 3ld Ed., 1999.
[00133] The term "alkaryl" or "'alkylaryl" refers to an alkyl group with an aryl substituent or an alkyl group linked to the molecule through an aryl group as defined herein. The term "aralkyl" or "arylalkyl" refers to an aryl group substituted with an alkyl substituent or linked to the molecule through an alkyl group as defined above. [00134] The term "cycloalkyl" includes a ring Of C3-8, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. [00135] The term "alkoxy" means a straight or branched chain alkyl group having an attached oxygen radical, the alkyl group having the number of carbons specified or any number within this range. For example, a "-O-alkyl'", C 1.4 alkoxy, methoxy, etc. [00136] The term "acyl"' or "'O-linked ester" includes a group of the formula C(O)R", wherein R' is an straight, branched, or cyclic alkyl (including lower alkyl), carboxylate residue of an amino acid, aryl including phenyl, heteroaryl, alkaryl, aralkyl including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl (including lower alkyl), aryl including phenyl optionally substituted with chloro, bromo, fluoro, iodo, Ci to C4 alkyl or Q to C4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxy-trityl, substituted benzyl, alkaryl, aralkyl including benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groups in the esters optimally comprise a phenyl group. In nonlimiting embodiments, acyl groups include acetyl, trifluoroacetyl, methylacetyl, cyclopropyl acetyl, cyclopropyl- carboxy, propionyl, butyryl, isobutyryl, hexanoyl, heptanoyloctanoyl, neo-heptanoyl, phenylacetyl, 2-acetoxy-2- phenylacetyl, diphenylacetyl, α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl, 2- nitro-benzeneacetyl, 4-chloro-benzeneacetyl, 2-chloro-2,2-diphenylacetyl, 2-chloro-2- phenylacetyl, trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl, bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl, chlorosulfonylacetyl, 3- methoxyphenylacetyl, phenoxyacetyl, tert-butyl acetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl, 7-chloro-dodecafluoro-heptanoyl, 7H- dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl,
53412 v2 DC 53 nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl, methyl 3-amino-5- phenylthiophene-2-carboxyl, 3,6-dichloro-2-methoxy-benzoyl, 4--(1 , 1 ,2,2-tetrafluoro-ethoxy)- benzoyl, 2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl, 3- cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl, pivaloyl acetyl, 1 -adamantane- carboxyl, cyclohexane-carboxyl, 2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane- carboxyl, perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolyl carbonyl, perfluorocyclohexyl carboxyl, crotonyl, l-methyl-lH-indazole-3-carbonyl, 2-propenyl, isovaleryl, 1 -pyrrolidinecarbonyl, 4-phenylbenzoyl. [00137] The term "acylamino" includes a group having a structure of "*-N(R')-C(O)-R", wherein each R is independently as defined above.
[00138] The term "carbonyl" includes a group of the structure "-C(O)-X-R" or "X-C(O)-R"', where X is O, S, or a bond, and each R is independently as defined above. [00139] The term "heteratom" includes an atom other than carbon or hydrogen in the structure of a heterocyclic compound, nonlimiting examples of which are nitrogen, oxygen, sulfur, phosphorus or boron.
[00140] The term "heterocycle", "heterocyclyl", or "heterocyclic" as used herein includes non-aromatic ring systems having four to fourteen members, preferably five to ten, in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom. Examples of heterocyclic rings include 3-l H-benzimidazol-2-one, (l -substituted)-2-oxo- benzimidazol-3-yl, 2-tetrahydro-furanyl, 3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3- tetrahydropyranyl, 4-tetra-hydropyranyl, [l ,3]-dioxalanyl, [l ,3]-dithiolanyl, [l ,3]-dioxanyl, 2- tetra-hydro-thiophenyl, 3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl, 4- morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2- pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1 -piperidinyl, 2-piperidinyl, 3- piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diazolonyl, N-substituted diazolonyl, 1- phthalimidinyl, benzoxanyl, benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, and benzothianyl. Also included within the scope of the term "heterocyclyl" or "heterocyclic", as it is used herein, is a group in which a non-aromatic heteroatom- containing ring is fused to one or more aromatic or non-aromatic rings, such as in an indolinyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the non-aromatic heteroatom-containing ring. The term "heterocycle", "heterocyclyl", or "heterocyclic" whether saturated or partially unsaturated, also refers to rings that are optionally substituted.
53412 v2'DC 54 (00141] The term "heteroaryl", used alone or as part of a larger moiety as in "heteroaralkyl" or "heteroaryl alkoxy", refers to heteroaromatic ring groups having five to fourteen members. Examples of heteroaryl rings include 2-furanyl, 3-furanyl, 3-furazanyl, N-imidazolyl, 2- imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3- pyiτolyl, 1-pyrazolyl, 2-pyi'azolyl, 3-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-pyriniidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2- triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, and benzoisoxazolyl. Also included within the scope of the term "heteroaryl", as it is used herein, is a group in which a heteroatomic ring is fused to one or more aromatic or nonaromatic rings where the radical or point of attachment is on the heteroaromatic ring. Examples include tetrahydroquinolinyl, tetrahydroisoquino-linyl, and pyrido [3,4-d]pyrimidinyl. The term "heteroaryl" also refers to rings that are optionally substituted. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic".
[001421 The term "amino"' as used herein unless otherwise specified, includes a moiety represented by the structure "-NR2", and includes primary, secondary and tertiary amines optionally substituted by alkyl, aryl, heterocyclyl, and/or sulfonyl groups. Thus R2 may represent two hydrogen atoms, two alkyl moieties, or one hydrogen and one alkyl moiety. [00143] The term "amido" as used herein includes an amino-substituted carbonyl, while the term "amidino" means a group having the structure "-C(NH)-NH2". [00144] The term "quaternary amine" as used herein includes quaternary ammonium salts that have a positively charged nitrogen. They are formed by the reaction between a basic nitrogen in the compound of interest and an appropriate quaternizing agent such as, for example, methyliodide or benzyliodide. Appropriate counterions accompanying a quaternary amine include acetate, trifluoroacetate, chloro, bromo and iodo ions. [00145] The term "substituted" includes multiple degrees of substitution by one or more named substituents such as, for example, halo, hydroxyl, thio, alkyl, alkenyl, alkynyl, nitro, cyano, azido, amino, carboxamido, etc. Where multiple substituent possibilities exist, the compound can be substituted by one or more of the disclosed or claimed substituent groups, independently from one another, and taken singly or plurally.
[00146] The term "protected" as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for
53412 v2< DC 55 other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
[00147] It should be understood that the various possible stereoisomers of the groups mentioned above and herein are within the meaning of the individual terms and examples, unless otherwise specified. As an illustrative example, "1 -methyl -butyl" exists in both (R) and the (S) form, thus, both (R)-I -methyl -butyl and (S)-I -methyl -butyl is covered by the term "1 -methyl-butyl", unless otherwise specified.
[00148] The term "biological sample", as used herein, includes, without limitation, cell cultures or extracts thereof; preparations of an enzyme suitable for in vitro assay; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.
[00149] The term "pharmaceutically acceptable carrier " refers to a non-toxic earner, adjuvant, or vehicle that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof. [00150] It should also be understood that when a carbon to carbon double bond group, such as C=C, or a carbon to nitrogen double bond group, such as C=N, has more than one substitents, the double bond geometry can be either E or Z. Both the E and Z forms of the double bond geometry fall within the scope of the present invention. Method of inhibiting the growth of'NF2 and NFl tumor cells [00151] The present invention includes methods of inhibiting the growth of NF2-deficient and/or NFl -deficient tumor cells by contacting, i.e., treating, the NF2-deficient or NFl - deficient tumor cells with radicicol and its derivatives, such as one or more compounds of formula I, II, III, IV, or V as described above. The present invention also includes methods of decreasing proliferation of NF2-deficient or NFl -deficient tumor cells by contacting the cells with radicicol and its derivatives, such as one or more compounds of formula I, II, III, IV, or V as described above.
[00152] In one embodiment, inhibition of the growth of NF2 or NFl cells is determined by comparing a sample of NF2-deficient or NFl -deficient tumor cells treated with radicicol and its derivatives to a control, such as a sample of untreated NF2-deficient or NFl -deficient tumor cells or a sample of cells treated with a known inert compound. Prior to contacting cells with radicicol and its derivatives, both samples of NF2-deficient cells or NFl-deficient cells (treated and control) should consist of approximately the same number of cells and be of the same cell type, e.g., NF2-deficient schwannomas or NFl -deficient MPNST cells. NF2- deficient tumor cells or NFl-deficient tumor cells treated with radicicol and its derivatives
53412 v2< DC 55 may decrease in number by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% following compound treatment compared to the control. [00153] Radicicol, a macrocyclic lactone antibiotic, has been shown to inhibit the function of HSP90. To further investigate the biological mechanism of radicicol and its analogs in regulating HSP90 and establish the fundamental structure-activity relationship, a number of radicicol analogs have been synthesized and studied. The term "radicicol analogs'" or "radicicol derivatives" as used herein denotes macrocyclic lactone compounds that are structurally similar to radicicol. Specifically, the "radicicol analogs'" or "radicicol derivatives"' refer to compounds of fused bicyclic ring structure wherein a six-membered aromatic ring shares two carbon atoms with a 12- to 16-membered non-aromatic ring containing a lactone group and at least one olefin group in the core of the 12- to 16-membered ring. The radicicol analogs/derivatives may have one or more substituents on the six- membered aromatic ring or the 12- to 16-membered non-aromatic ring. It is noted that the terms "analog" and "derivative" are used interchangeably in the present application.
[00154] A number of radicicol analogs have been disclosed in patent publications including WO 96/33989, WO 98/18780, WO 99/55689, US 7,1 15,651, US 5,731,343, and US 5,077, 165, all of which are herein incorporated by reference in their entirety. Radicicol and its representative analogs are shown in Scheme IA. [00155] SCHEME I A
Figure imgf000058_0001
Figure imgf000058_0002
Cyclopropane Radicicol
of Radicicol
(compound 9b, Ikuina, 2003. J. Med. Chem., 46. 2534)
53412 \2 DC 57 [00156] In one embodiment of the present invention, radicicol and derivatives thereof have a structure of formula I, II, III, IV, or V as described above.
[00157] In another embodiment of the present invention, radicicol and derivatives thereof are used in combination with at least one additinoal active agent to inhibit the growth of NF2- deficient and/or NFl -deficient tumor cells. Preferably, the at least one additional active agent is one or more HSP90 inhibitors. That is, the methods of inhibiting the growth of NF2- deficient and/or NFl -deficient tumor cells further comprises contacting, i.e., treating, the NF2-deficient or NFl -deficient tumor cells with at least one additinoal active agent, such as one or more HSP90 inhibitors. In one embodiment, the compound of the present invention contacts the NF2-deficient or NF 1 -deficient tumor cells with another active agent. The term "active agent" as used herein refers to a chemical or biological entity that can be used, for example, as a therapeutic. Active agents can also be used to mitigate toxicity. For instance, a PAK inhibitor can be used in conjunction with a compound of the present invention to inhibit cell growth or cause cell death. [00158] HSP90 functions as a chaperone for its client proteins as part of dynamic multiprotein complexes, here define as "ΗSP90 complexes" or "'HSP90 chaperone machineries, which consist of co-chaperones such as HSP90, HSP70, HSP27, HSP40, HOP, p23, and CDC37 and individual client protein. These multimeric chaperone/co-chaperone complexes are quite dynamic and involve a series of cycles of association and dissociation of several co-chaperones with HSP90 and/or client proteins.
[00159] As used herein, "a HSP90 inhibitor," "a HSP90 inhibitor compound," "a HSP inhibitory compound," "'an inhibitor compound", or "an inhibitor"' refers to a substance that inhibits or reduces the growth of or number of NF2-deficient tumor cells or NFl -deficient tumor cells by inhibiting HSP90 function in these cells. Any compound that modulates the activity of HSP90, i.e., a HSP90 modulator, may also be used to inhibit the growth of NF2- deficient tumor cells. For example, a HSP90 modulator may modulate HSP90 by inhibiting or down-regulating, i.e., reducing, the activity of one or more proteins of the HSP90 complex. [00160] In one embodiment of the present invention, biological agents or molecules that inhibit or reduce the activity of the HSP90 complex may also be used to inhibit the growth of NF2-deficient or NFl -deficient tumor cells. Examples of the biological agents or molecules of the present invention, include, but are not limited to antibodies, peptides, siRNAs, antisense nucleic acids, and any combinations thereof.
[00161] Inhibition by a HSP90 inhibitor can occur through a reduction in the capability of the HSP90 protein or complex to assist its client proteins to function in or to activate a signaling
53412 DC 5g pathway. The HSP90 inhibitor may reduce the amount of total or phosphorylated forms of specific client proteins of HSP90 which in turn inhibits the activity of the downstream pathways of these client proteins. The HSP90 inhibitor/modulator can upregulate the amount of the HSP70 protein and other HSP proteins in the HSP90 complex. HSP90 function can be inhibited or modulated, for example, such as by binding of a compound as disclosed herein to HSP90 or by modifying the HSP90 protein postranslationally. Also, such as by inhibition of a kinase activity of a protein, by inhibition of dimerization of a protein, inhibition of DNA binding of a protein, or inhibition of transactivation of a protein. HSP90 inhibitors can inhibit one or more proteins in the HSP90 complex by an antibody specific for it or by employing antisense, siRNA, or ribozyme technologies to reduce the level of mRNA coding for the protein.
[00162] The HSP90 inhibitors or modulators useful for the present invention include, but are not limited to benzoquinone-based ansamycin antibiotics, purine scaffold-based HSP90 inhibitors, pyrazole or imidazole scaffold-based HSP90 inhibitors, tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitors, novobiocin and its analogs, and any combinations thereof.
[00163] One well-known class of HSP90 inhibitors are benzoquinone-based ansamycin antibiotics, the structure of which typically contain a 1 ,4-benzoquinone ring fused with a 18- to 20-membered non-aromatic ring containing a lactam group and at least one olefin group in the core of the 18- to 20-membered ring. (See, for example, WO 98/51702, which is herein incorporated by reference in its entirety) Some representative benzoquinone-based ansamycin antibiotics are shown in Scheme 2A. [00164] SCHEME 2A
Figure imgf000060_0001
Geldanamycin: R' = H, R1 = H, RJ = OCH3 Dihydroquinone derivative of 17-AAG Herbimycin: R1 = CH3, R2 = OCH3, R3 = H 17-AAG: R1 = H, R2 = H, R3 = -NHCH2CH=CH2 17-DMAG: R1 = H, R2 = H, R3 = -NHCH2CH2N(CH3),
53412 v2 DC 59 [00165] It has been reported that certain purine scaffold-based compounds are HSP90 inhibitors. (See, for example, WO 02/36705, WO 03/037860, and WO 2006/084030, all of which are herein incorporated by reference in their entirety) These purine scaffold-based HSP90 inhibitors typically have a structure wherein an adenine ring and a six-membered aryl or heteroaryl ring are linked through a linker which can be methylene, fluorinated methylene, sulfur, oxygen, nitrogen, carbonyl, imine, sulfmyl, or sulfonyl. Scheme 3A shows two compounds exemplifying the purine scaffold-based HSP90 inhibitors. [00166] SCHEME 3A
Figure imgf000061_0001
[00167] Some pyrazole or imidazole scaffold-based compounds are known to inhibit HSP90. These pyrazole or imidazole scaffold-based HSP90 inhibitors are typically non-fused tricyclic compounds wherein two aryl or heteroaryl rings are attached to two adjacent positions (carbon or nitrogen atom) of a pyrazole or imidazole ring, respectively. (See, for example, WO 2007/021877, which is herein incorporated by reference in its entirety, or Vernalis Ltd, Bioorg Med Chem Lett, 2006, 16, 2543-2548, or Sharp et al., Molecular Cancer Therapeutics, 2007, 6, 1 198-121 1). Examples of pyrazole or imidazole scaffold-based HSP90 inhibitors are shown in Scheme 4A. [00168] SCHEME 4A
Figure imgf000061_0002
CCT018159 CCT0129397 VER49009
53412 v2/DC 60
Figure imgf000062_0001
Compound 1 in Table 5 Compound 2 in Table 5 Compound 3 in Table 5
WO 2007/021877 WO 2007/021877 WO 2007/021877
[00169] Another class of HSP90 inhibitors are tetrahydroindolone and tetrahydroindazolone derivatives reported in WO 2006/091963, the disclosure of which is herein incorporated by reference in its entirety. These tetrahydroindolone or tetrahydroindazolone based HSP90 inhibitors generally have a structure wherein a substituted aryl group is directly attached to the nitrogen atom of a tetrahydroindolone or tetrahydroindazolone. Scheme 5 A shows certain examples in WO 2006/091963. [00170] SCHEME 5A
Figure imgf000062_0002
Example 3 Example 11
WO 2006/091963 WO 2006/091963
[00171] The aminoglycoside antibiotic, Novobiocin, has also been reported to inhibit HSP90. (See, for example, Yu et al, J. Am. Chem. Soc, 2005, 127, 12778-12779) The structure of Novobiocin is shown in Scheme 6A. [00172] SCHEME 6A
Figure imgf000062_0003
53412 v2 DC 61 [00173] Many client proteins of HSP90 are kinases which are involved in phosphorylation cascades. For this reason, presence and/or activity of a client protein of HSP90 can be assessed by measuring the phosphorylation of the protein and/or a downstream protein. Inhibition of phosphorylation or hypophosphorylation is an indication of a decrease or inhibition of activity of a protein. In one embodiment, the proliferation of NF2-deficient tumor cells or NFl -deficient cells can be reduced or stabilized by the administration of a HSP90 inhibitory compound that inhibits or reduces the phosphorylation of one or more proteins of the PI3K/Akt pathway or Ras-Raf pathway including, but not limited to, Akt, mTOR, FKHR, GSK3, S6, S6K, Bad, Raf, Mek, and Erkl/2 proteins. [00174] In another embodiment of the invention, the proliferation of NF2-deficient or NFl- deficient tumor cells can be reduced or stabilized by the administration of a compound of the present invention and/or a HSP90 inhibitory compound that reduces or degrades ErbB2 or phospho-ErbB2. [00175] In another embodiment of the invention, the proliferation of NF2-deficient or NFl - deficient tumor cells can be reduced or stabilized by the administration of a compound of the present invention and/or a HSP90 inhibitory compound that reduces or degrades other receptor tyrosine kinases.
[00176] A cytotoxic agent and a compound of the present invention can also be contacted together with NF2-deficient or NFl -deficient tumor cells, either simultaneously or sequentially. In this embodiment, the cytotoxic agent, i.e., chemotherapeutic drug, can act synergistically with the HSP90 inhibitor compound to inhibit growth and kill NF2-deficient or NFl -deficient tumor cells. Cytotoxic agents that can be co-administered with the compound of the present invention are chemotherapy drugs known in the art. [00177] Growth of NF2-deficient or NFl -deficient tumor cells can be inhibited in vitro or ex vivo. NF2-deficient or NFl -deficient tumor cells can be primary cell culture cells or a tissue culture cell line cells. Cells can be mammalian cells, including, but not limited to, human, canine, rat and murine yV/2-deficient or JV/7 -deficient tumor cells. For instance, methods of the invention include the use of Λf/2-deficient mouse cells {e.g., Schwann cells) or NF2- deficient human tumor cells {e.g., Schwann cells) or NfI -deficient mouse cells {e.g. Schwann cells and MPNST cells) or NFl -deficient human tumor cells (e.g. MPNST cells, astrocytoma cell, JMML cells). Cells can be confluent or subconfluent when contacted with radicico or its derivatives.
[00178] In another embodiment, NF2-deficient or NFl -deficient tumor cells are contacted with radicicol or its derivatives in vivo. In this embodiment, radicicol or its derivatives is
53412 v2' DC 52 administered to a subject with a NF2-deficient or NFl -deficient tumor. The subject can be any mammal, including, but not limited to, a human, canine, rat, mouse, and farm animals. Radicicol or its derivatives can be administered at the site of a NF2-deficient or NFl -deficient tumor or elsewhere in the body. [00179] NF2-deficient or NFl -deficient tumor cells can be contacted in vitro or ex vivo with very small amounts of compound of the present invention to trigger inhibition of cell growth and/or resulting in cell death. For instance, NF2-deficient or NFl -deficient cells can be contacted with at least about .001 μM of compound of the present invention, at least about .01 μM of compound of the present invention, at least about .05 μM of compound of the present invention, at least about 0.08 μM of compound of the present invention, at least about 0.09 μM of compound of the present invention, at least about 0.1 μM of compound of the present invention, at least about 0.2 μM of compound of the present invention, at least about 0.3 μM of compound of the present invention, at least about 0.4 μM of compound of the present invention, at least about 0.5 μM of compound of the present invention, at least about 0.6 μM of compound of the present invention, at least about 0.7 μM of compound of the present invention, at least about 0.8 μM of compound of the present invention, at least about 0.9 μM of compound of the present invention, at least about 1 μM of compound of the present invention, at least about 1.5 μM of compound of the present invention, at least about 2 μM of compound of the present invention or about 2 μM to 10 μM of compound of the present invention to inhibit cell growth and/or trigger cell apoptosis. Appropriate amounts of the compound to contact with the cells can be determined by persons skilled in the art to obtain the desired level of inhibition of cell growth or the corresponding amount of cell death. [00180] Inhibition of cell growth or cell death can be measured by methods known in the art. For instance, in vitro and ex vivo inhibition of cell growth and cell death can be measured by a cell proliferation assay and a cell apoptosis assay. Cell death or inhibition of proliferation can be measured in terms of the IC50 of the compound of the present invention. In one embodiment, the IC50 of NF2-deficient tumor cells treated in vitro or ex vivo is at least about .001 μM, at least about .01 μM, at least about .05 μM, at least about 0.08 μM, at least about 0.09 μM, at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, at least about 0.8 μM, at least about 0.9 μM, at least about 1 μM, at least about 1.5 μM, at least about 2 μM, or about 2 μM to 10 μM compound of the present invention.
[00181] In another embodiment, NF2-deficient or NFl -deficient tumor cells are contacted with a compound of the present invention in vivo. In this embodiment, a compound of the
53412 v2 DC 63 present invention is administered to a subject with a NF2-deficient or NFl -deficient tumor. The subject can be any mammal, including, but not limited to, a human, canine, rat, mouse, and farm animals. The compound can be administered at the site of the tumor or elsewhere in the body. [00182] When a compound of the present invention is administered in vivo, it can be administered such that the serum levels mimic those levels found to cause inhibition of cell growth and/or cell death in vitro, In one embodiment, the compound of the present invention contacts the NF2-deficient or NFl -deficient tumor cells with another agent. Methods of Treatment [00183] The present invention provides methods of treating a subject diagnosed with neurofibromatosis type-2 or a condition associated with the loss of NF2 function (i.e., the loss of the protein, Merlin), including, but not limited to sporadic schwannomas, and other NF2 related tumors, such as sporadic meningiomas and sporadic mesotheliomas. The method comprises administering to a patient a therapeutically effective amount of one or more compounds of the present invention. "Subject'" and "'patient" are used interchangeably herein, and refer to a mammalian subject to be treated, including, but not limited to, human patients. A subject can be diagnosed with NF2 by methods known in the art, including, but not limited to, a direct gene test and the bilateral occurrence of vestibular schwannomas. The methods of treatment are similar for a subject diagnosed with NFl or a condition associated with the loss of NFl function (i.e., the loss of the protein, neurofibromin).
[00184] The present invention also provides methods of treating a subject diagnosed with a neurodegenerative disease. A neurodegenerative disease is a central nervous system disorder characterized by gradual and progressive loss of neural tissue. Neurodegenerative diseases result from deterioration of neurons which over time will lead to neurodegeneration and disabilities resulting from this. Examples of neurodegenerative diseases include, but are not limited to Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease, Huntington disease, multiple sclerosis, Parkinson disease, and spinal muscular atrophy but other neurodegenerative diseases are known to persons skilled in the field. [00185] As used herein, "therapeutically effective amount" refers to an amount sufficient to inhibit the growth of one or more NF-2 deficient tumors or NFl -deficient tumors, or to slow, stop, or reverse the gradual and progressive loss of neural tissue. "Inhibition", as used herein, means that the size of a tumor stabilizes and does not increase. In one embodiment, the tumor may be reduced in size following administration of a compound of the present invention. For instance, the tumor may be reduced in size at least about 10%, at least about
53412 v2'DC 64 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the size of the tumor prior to treatment, i.e., a baseline measurement. The inhibition of tumor growth can be assessed by methods known in the art, including, but not limited to, magnetic resonance imaging or a CAT scan.
[00186] As can be appreciated by a skilled artisan, a "therapeutically effective amount" will vary depending on the tumor load of the patient and the age, weight, and other conditions of the patient to be treated. Further, a therapeutically effective amount may vary based on the specific compound of the present invention administered and pharmaceutical composition as well as the route of administration.
[00187] The methods of the present invention can be used to treat a variety of NF2-deficient tumors or NFl -deficient tumors. For instance, the compound of the present invention can be administered to treat a vestibular schwannoma, for instance, a unilateral schwannoma or a bilateral schwannoma. Inhibition of the growth of these tumors and/or death of tumor cells can mitigate the symptoms associated with schwannomas, including but not limited to vestibular schwannomas. For instance, treatment with a compound of the present invention may result in an improvement in hearing, tinnitus and/or balance. [00188] The methods of the invention can be used to treat other types of NF2-deficient schwannomas as well or type of NFl -deficient tumors. For instance, administration of a compound of the present invention can be used to treat a patient suffering from one or more spinal cord schwannomas/neurofibromas and other peripheral nerve schwannomas/neurofibromas and sporadic schwannomas/neurofibromas. Treatment of peripheral nerve tumors, including spinal cord tumors, can decrease a patient's pain. Further, neurological deficits associated with NFl may be reversible with the treatment of the present invention.
[00189] In one embodiment of the invention, administration of a compound of the present invention results in a decrease in a patient's tumor load. For instance, administration of a a compound of the present invention can result in at least about a 5%, at least about a 10%, at least about a 15%, at least about a 20%, at least about a 25%, at least about a 30%, at least about a 40%, at least about a 50%, at least about a 60%, at least about a 70%, at least about an 80%, at least about a 90%, or about a 100% decrease in tumor load. Pharmaceutical Compositions
[00190] The compound of the present invention may be formulated for administration in a pharmaceutically acceptable earner or excipient in accordance with known techniques in the
53412 v2/DC 55 art. See, e.g., Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical composition according to the invention, the compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the composition and must not be deleterious to the patient. The earner may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for instance, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of compound. In one embodiment of the invention, the pharmaceutical composition administered to the patient contains more than one type of HSP90 inhibitor.
[00191] Pharmaceutical compositions include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, etc.), topical (e.g., both skin and mucosal surfaces, including airway surfaces) and transdermal administration. [00192] In one embodiment of the invention, the pharmaceutical composition is applied directly to the site of a NF2-deficient tumor or NFl -deficient tumor. For instance, the HSP90 inhibitor can be applied by local treatment which encompasses both topical treatment and intralesional or intradermal treatment at the site of the tumor. Therefore, the inhibitor can be injected into, topically applied onto or near a NF2-deficient tumor or NFl-deficient tumor. In one embodiment of the invention, the inhibitor is applied intralesionally to NF2-deficient tumors or NF l -deficient tumors by methods known in the art.
[00193] Alternatively, the pharmaceutical composition can take the form of an implant. Such a composition can be surgically implanted at or near the site of a tumor for slow release of the compound of the present invention. [00194] In another embodiment of the invention, a pharmaceutical composition is administered by transcatheter arterial embolization by methods known in the art. [00195] Pharmaceutical compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a compound of the present invention; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
[00196] Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or
53412 v2 'DC 65 tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
[00197] Pharmaceutical compositions of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. [00198] In one embodiment of the invention, a pharmaceutical composition of the compound of the present invention is applied topically. For instance, a phaπnaceutical composition can be applied to the skin near a NF2-deficient tumor or a NFl -deficient tumor. Compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
[00199] Pharmaceutical compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such compositions may be applied near or at the site of a NF2-deficient tumor or a NFl -deficient tumor. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient. [00200] The present invention also includes methods of treating a subject with NF2 or NFl by administering a liposomal formulation of a compound of the present invention. The technology for forming liposomal suspensions is well known in the art. When the inhibitor is water-insoluble, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. The liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques. The liposomal formulations containing the compound of the present invention disclosed herein or salts thereof, may also be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
53412 v2< DC 67 [00201] Other pharmaceutical compositions, such as aqueous base emulsions, may be prepared from compounds of the present invention. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of compounds of the present invention. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.
[00202] The therapeutically effective dosage of any specific compound of the present invention for treatment of a NF2-deficient tumor or a NF 1 -deficient tumor will vary from patient to patient, and will depend upon factors such as the age, weight, gender and condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.01 or 0.1 to about 50, 100 or 500 mg/kg will have therapeutic efficacy, with all weights being calculated based upon the weight of the compound, including the cases where a salt is employed.
[00203] Mammals, and specifically humans, suffering from a respiratory disorder can be treated by the inhalation, systemic, oral, topical, or transdermal administration of a composition comprising an effective amount of the compounds described herein or a pharmaceutically acceptable salt, ester or prodrug thereof, optionally in a pharmaceutically acceptable carrier or diluent.
[00204] In a separate embodiment, the compounds of the invention are in the form of an inhaled dosage. In this embodiment, the compounds may be in the form of an aerosol suspension, a dry powder or liquid particle form. The compounds may be prepared for delivery as a nasal spray or in an inhaler, such as a metered dose inhaler. Pressurized metered-dose inhalers ("MDF") generally deliver aerosolized particles suspended in chlorofluorocarbon propellants such as CFC-1 1, CFC- 12, or the non-chlorofluorocarbons or alternate propellants such as the fluorocarbons, HFC- 134A or HFC-227 with or without surfactants and suitable bridging agents. Dry-powder inhalers can also be used, either breath activated or delivered by air or gas pressure such as the dry-powder inhaler disclosed in the Schering Corporation International Patent Application No. PCT/US92/05225, published 7 Jan. 1993 as well as the Turbuhaler™ (available from Astra Pharmaceutical Products, Inc.) or the Rotahaler™ (available from Allen & Hanburys) which may be used to deliver the aerosolized particles as a finely milled powder in large aggregates either alone or in combination with some pharmaceutically acceptable earner e.g. lactose; and nebulizers. [00205] The compounds of the invention may be also administered in specific, measured amounts in the form of an aqueous suspension by use of a pump spray bottle. The aqueous suspension compositions of the present invention may be prepared by admixing the
53412 v2, DC 68 compounds with water and other pharmaceutically acceptable excipients. The aqueous suspension compositions according to the present invention may contain, inter alia, water, auxiliaries and/or one or more of the excipients, such as: suspending agents, e.g., microcrystalline cellulose, sodium carboxymethylcellulose, hydroxpropyl-methyl cellulose; humectants, e.g. glycerin and propylene glycol; acids, bases or buffer substances for adjusting the pH, e.g., citric acid, sodium citrate, phosphoric acid, sodium phospate as well as mixtures of citrate and phosphate buffers; surfactants, e.g. Polysorbate 80; and antimicrobial preservatives, e.g., benzalkonium chloride, phenylethyl alcohol and potassium sorbate. [00206] Typical systemic dosages for all of the herein described conditions are those ranging from 0.01 mg/kg to 1500 mg/kg of body weight per day as a single daily dose or divided daily doses. Preferred dosages for the described conditions range from 0.5-1500 mg per day. A more particularly preferred dosage for the desired conditions ranges from 5-750 mg per day. Typical dosages can also range from 0.01 to 1500, 0.02 to 1000, 0.2 to 500, 0.02 to 200, 0.05 to 100, 0.05 to 50, 0.075 to 50, 0.1 to 50, 0.5 to 50, 1 to 50, 2 to 50, 5 to 50, 10 to 50, 25 to 50, 25 to 75, 25 to 100, 100 to 150, or 150 or more mg/kg/day, as a single daily dose or divided daily doses. In one embodiment, the compounds are given in doses of between about 1 to about 5, about 5 to about 10, about 10 to about 25 or about 25 to about 50 mg/kg. Typical dosages for topical application are those ranging from 0.001 to 100% by weight of the active compound. [00207] The compounds are conveniently administered in units of any suitable dosage form, including but not limited to one containing from about 7 to 3000 mg, from about 70 to 1400 mg, or from about 25 to 1000 mg of active ingredient per unit dosage form. For example, an oral dosage of from about 50 to 1000 mg is usually convenient, including in one or multiple dosage forms of 50, 100, 200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 mgs. Lower dosages may be preferable, for example, from about 10-100 or 1 -50 mgs. Also contemplated are doses of 0.1 -50 mg, 0.1 -20 mgs., or 0.1-10 mgs. Furthermore, lower doses may be utilized in the case of administration by a non-oral route, as for example, by injection or inhalation. [00208] The compound is administered for a sufficient time period to alleviate the undesired symptoms and the clinical signs associated with the condition being treated.
[00209] The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutic amount of compound in vivo in the absence of serious toxic effects. Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions are generally known in the art. They include, but
53412 v2 DC 59 are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, solvents, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silicates, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, oils, polyemylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. Pharmaceutically accepted vehicles can contain mixtures of more than one excipient in which the components and the ratios can be selected to optimize desired characteristics of the formulation including but not limited to shelf-life, stability, drug load, site of delivery, dissolution rate, self-emulsification, control of release rate and site of release, and metabolism.
[00210] Formulations can be prepared by a variety of techniques known in the art. Examples of formulation techniques can be found in literature publications and in texts such as "Water- insoluble drug formulation", edited by Rong Liu, 2000, Interpharm Press.
[00211 ] If administered intravenously, carriers can be physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other surface- active emulsifying agents or bioavailability enhancers which are commonly used in the
53412 v2/DC 70 manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
[00212] The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
[00213] One mode of administration of the active compound for systemic delivery is oral. Oral compositions will generally include an inert diluent or an edible earner. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. [00214] The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [00215] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. [00216] The compound or its salts can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
[00217] In a preferred embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release
53412 v2-'DC 71 formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable earners. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
[00218] Suitable vehicles or earners for topical application can be prepared by conventional techniques, such as lotions, suspensions, ointments, creams, gels, tinctures, sprays, powders, pastes, slow-release transdermal patches, suppositories for application to rectal, vaginal, nasal or oral mucosa. In addition to the other materials listed above for systemic administration, thickening agents, emollients, and stabilizers can be used to prepare topical compositions. Examples of thickening agents include petrolatum, beeswax, xanthan gum, or polyethylene, humectants such as sorbitol, emollients such as mineral oil, lanolin and its derivatives, or squalene. Combination Therapy
[00219] The compound of present invention can also be mixed with other active agents which do not impair the desired action, or with materials that supplement the desired action. The one or more active agents can be administered in conjunction, i.e. combination or alternation, with other medications used in the treatment of disorders or conditions associated with NFl or NF2. The term "active agent" has the same meaning as described above. In other words, methods of the invention include co-administering a compound of the present invention with another therapeutic agent, either concurrently or sequentially. Preferably, the compounds of the present invention can be administered in combination or alternation with one or more HSP90 inhibitors as described above.
534)2 v2 DC 72 [00220] In one embodiment, a compound of the present invention can be administered with a cytotoxic agent known in the art to inhibit tumor growth or kill the tumor(s). Coadministration of a HSP90 pathway inhibitor with a cytotoxic agent may reduce the toxic side-effects otherwise associated with the cytotoxic agent. Toxicity would be reduced because less cytotoxic agent would need to be administered to the patient to inhibit tumor growth or kill one or more tumors than if the cytotoxic agent were administered alone. [00221] In addition to co-administration of the compound of the present invention with a cytotoxic agent such a chemotherapy drug, the compound of the present invention can also be administered with radiation therapy in combination. As with chemotherapy, administration of the compound of the present invention with radiation therapy allows less radiation therapy to be used for treatment, and thus, reduces the risk of toxic side effects. [00222] The compound of the present invention can also be administered before or after surgical removal of a tumor to kill or inhibit the growth of any remaining NF2-deficient or NFl -deficient tumor cells. This embodiment is especially useful for tumors of the brain and spine where removing all NF2-deficient or NFl -deficient tumor cells could put the patient at risk for permanent nerve damage or even death.
[00223] In another embodiment, the compound of the present invention is administered to a patient with a PAK inhibitor. [00224] It is also possible to combine any compound of the invention with one or more other active agents in a unitary dosage form for simultaneous or sequential administration to a patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. [00225] Co-administration of a compound of the invention with one or more other active agents generally refers to simultaneous or sequential administration of a compound of the invention and one or more other active agents, such that therapeutically effective amounts of the compound of the invention and one or more other active agents are both present in the body of the patient. [00226] Co-administration includes administration of unit dosages of the compounds of the invention before or after administration of unit dosages of one or more other active agents, for example, administration of the compounds of the invention within seconds, minutes, or hours of the administration of one or more other active agents. For example, a unit dose of a compound of the invention can be administered first, followed within seconds or minutes by administration of a unit dose of one or more other active agents. Alternatively, a unit dose of
53412 v-2/DC 73 one or more other active agents can be administered first, followed by administration of a unit dose of a compound of the invention within seconds or minutes. In some cases, it may be desirable to administer a unit dose of a compound of the invention first, followed, after a period of hours (e.g., 1 -12 hours), by administration of a unit dose of one or more other active agents. In other cases, it may be desirable to administer a unit dose of one or more other active agents first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a compound of the invention.
[00227] The combination therapy may provide "synergy" and "synergistic effect", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1 ) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., in separate tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. Methods of Screening for NF2 and NFl HSP90 Inhibitor Therapeutics [00228] The present invention also includes methods of screening HSP90 inhibiting compounds for efficacious treatment of NF2 or NFl . For instance, the methods of the invention can be used to determine the efficacy of a compound in pre-clinical experiments. This is useful because it can allow researchers to reduce the number of drugs to be tested for efficacious treatment of NF2 or NFl . The methods of the invention can also be used to repurpose existing drugs that were developed and/or are used for other indications. This is useful because such drugs presumably have established safety data and can thus be brought to market quicker and with less expense than uncharacterized compounds. [00229] As used herein, "efficacious treatment" means that a test compound results in the inhibition or reduction of cell growth. In one embodiment, efficacious treatment results in cell death or inhibition of proliferation. An efficacious treatment can be correlated with a reduction in the amount or activity of one or more client proteins of HSP90 or an increase in other HSP proteins such as HSP70. For instance, a reduction in the amount of ErbB2 protein leads to the reduction in the amount of phospho-ErbB2 and the activity of ErbB2 which can be correlated with the inhibition or reduction of growth of NF2-deficient cells. In addition, a
53412 v2/ DC 74 reduction in the amount of AKT or phospho-AKT may lead to the reduction in the phosphorylation of mTOR, GSK3, FKHR, S6 and S6K which can also be correlated with the inhibition or reduction of growth of NF2-deficient cells or apoptosis of NF2-deficient cells. Moreover, a reduction in the amount of Raf may lead to the reduction in the activity of downstream MAP kinase signaling pathway which can be correlated with the inhibition or reduction of growth of NFl -deficient cells.
[00230] Methods known in the art can be used to assess whether a test drug reduces the activity of one or more client proteins of the HSP90 complex. Such assays can be conducted in vitro, ex vitro using cells or tumor specimens from human patients or animal models, or, in the case of animal models, in vivo (Roe et al., 2004, Cell 1 16: 87-98; Smith et al., 2005,
Cancer Chemother. Pharmacol. 56: 126-137; Banerji et al., 2005, Clin. Cancer Res. 1 1 :7023- 7032; Eiseman et al., 2005, Cancer Chemother. Pharmacol. 55: 21-32; Goetz et al, 2005, JL Clin. Oncol. 23(6): 1078-1087; Solit et al., 2002, Clin. Cancer. Res. 8(5):986-993) [00231] In one embodiment of the invention, the ability of a drug to reduce the activity of a client protein (e.g. a kinase) of HSP90 or the downstream pathway of the client protein is assessed by contacting the proteins of the cells with an anti-phospho antibody specific for the particular protein. For instance, Western blots can be used by methods known in the art and can be probed with anti-phospho antibodies to phosphorylated AKT, phosphorylated ErbB2, phosphorylated GSK3, phosphorylated S6, phosphorylated S6K, phosphorylated mTOR, phosphorylated Mek, and phosphorylated down-stream proteins thereof. [00232] Embodiments of the invention are summarized as follows:
[00233] The present invention is directed to a method of treating, preventing or ameliorating tumors or symptoms resulting from neurofibromatosis in a subject comprising administering to said subject with neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or with neurofibromatosis type 1 (NFl) or a condition associated with the loss of NFl function a therapeutically effective amount of at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor which inhibits or slows growth of one or more NF2-deficient tumors or NFl -deficient tumors, reduces the number of said tumors or inhibits and/or reduces associated symptoms as compared to no treatment with the composition as a control level to determine treatment utility. The method is particularly directed to the administration of the at least one composition comprising HSP90 resulting in a decrease in size and/or number of one or more NF2-deficient tumors or of one or more NFl- deficient tumors.
53412 v2 DC 75 [00234] The method of the present invention comprises administering a HSP90 inhibitor comprising a compound which inhibits or reduces the function of HSP90 complex. More specifically, the HSP90 inhibitor comprises a compound that binds and inhibits the HSP90 protein, modifies HSP90 protein posttranslationally, and/or increases HSP70 or other HSP proteins from their normal levels in the NF2-deficient or NFl -deficient tumors. The administration of the HSP90 inhibitor results in the upregulation or increase in HSP70 in said subject. Further, the method comprises administering a HSP90 inhibitor comprising a compound that degrades or reduces one or more client proteins of HSP90 and the phosphorylated forms of the client proteins. Additionally, the method comprises administering a HSP90 inhibitor comprises a compound that inhibits or reduces activity or phosphorylation of signaling pathway proteins associated with one or more client proteins of HSP90. The one or more client proteins are selected from the group consisting of ErbB2, AKT, and Raf. The HSP90 inhibitor inhibits or reduces activity or phosphorylation of the signaling pathways proteins, such as PDK, mTOR, GSK3, 4E-BP1 , Bad, FKHR, HSP90, S6K, S6, Mek, and Erkl/2.
[00235] In a further embodiment, the method comprises administering a HSP90 inhibitor comprising a biological agent or molecule that is a peptide, antibody, siRNA and antisense nucleic acid molecule. [00236] In still a further embodiment, the method comprises administering at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor comprising radicicol or a derivative thereof, 17-AAG or a derivative thereof, a purine scaffold-based HSP90 inhibitor or a derivative thereof, a pyrazole scaffold-based HSP90 inhibitor or a derivative thereof, an imidazole scaffold-based HSP90 inhibitor or a derivative thereof, a tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitor or a derivative thereof, Novobiocin or a derivative thereof, or any combination thereof. Further, in a further embodiment, the HSP90 inhibitor comprises a HSP90 modulator compound such as a HDAC inhibitor and specifically HDAC6 inhibitor. This HSP90 modulator compound may further comprise Trichostatin A, SAHA, LAQ824, FK228, or derivatives thereof. Further, the method comprises administering a HSP90 inhibitor comprising an inhibitor or compound that modulates or increases the level of HSPs such as HSP70 and HSP27. The HSP90 inhibitor further comprises NZ28 (NCS-134754) or a derivative thereof.
[00237] The method comprises administering the HSP90 inhibitor orally, intravenously or locally, such as intralesionally or topically. The method also encompasses administering the HSP90 inhibitor before, during or after surgical removal of a tumor, Another mode of
53412 v2/DC 76 administration allows the HSP90 inhibitor to be co-administered before, during or after radiation therapy or with a PAK inhibitor or a cytotoxic compound. [00238] The method of the present invention treats, prevents or ameliorates tumors or symptoms resulting from neurofibromatosis type 2 (NF2) or condition(s) associated with the loss of NF2 function. More specifically the one or more NF2-deficient tumors comprise vestibular schwannomas, and more specifically comprise a unilateral vestibular schwannoma or a bilateral vestibular schwannoma. Additionally, the one or more NF2-deficient tumors that are treated comprise spinal cord schwannomas, sporadic schwannomas, peripheral nerve schwannomas, schwannoma, meningioma, mesothelioma, ependymoma, glioma and astrocytoma.
[00239] The method comprises the administration of the HSP90 inhibitor to obtain results in an improvement in at least one of the subject's hearing, balance and vision; increase in muscle mass, reduction in tumor burden in the subject, which the latter is identified using a MRI or a CAT scan. [00240] The method of the present invention treats, prevents or ameliorates tumors or symptoms resulting from neurofibromatosis type 1 (NFl) or condition(s) associated with the loss of NFl function. More specifically the one or more NFl -deficient tumors comprise a dermal and plexiform neurofibromas, optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ependymomas, pheochromocytomas and ganglioneuromas, rhabdomyosarcomas, neurofibrosarcomas, malignant peripheral nerve sheath tumors ("MPNST"), malignant schwannomas, and JMML.
[00241] The present invention also includes a method of inhibiting or reducing the growth or number of NF2-deficient tumor cells or NFl -deficient tumor cells comprising contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor which inhibits or slows growth and/or reduces the number of one or more NF2-deficient tumors or NFl -deficient tumors. This method comprises contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with said compound occurs in vitro or ex vivo. The NF2-deficient tumor cells are Nf2-deficient mouse Schwann cells and said NFl -deficient tumor cells are Nf 1 -deficient mouse Schwann cells. Or the NF2-deficient tumor cells are NF2-deficient human schwannoma cells and said NFl -deficient tumor cells are NFl -deficient human Schwann cells. The NF2-deficient tumor cells are selected from the group consisting of NF2-deficient schwannoma cell line cells, NF2-deficient meningioma cell line cells and NF2-deficient mesothelioma cell line cells. The NF2-deficient tumor cells are selected from the group
53412 v2 DC 77 consisting of HEI193 cells, SF1335 cells, BAR cells and RAV cells. The NFl -deficient tumor cells are selected from the group consisting of human MPNST cells, primary neurofibroma cells derived from NFl patients, mouse N/Z/pJJ-deficient MPNST cell lines established from cisNfl ;p53 mice, and NfI-/- mouse cells, such as Schwann cells, mouse embryonic cells, and leukemia cells. More specifically, the NFl -deficient tumor cells are selected from the group consisting of ST88-14, 88-3, 90-8, and sNF96.2. The NF2-deficient tumor cells or NFl- deficient tumor cells that are contacted with said HSP90 inhibitor can occur in vivo. The NF2-deficient tumor cells or said NFl -deficient tumor cells are from a human, canine, rat or mouse. [00242] The method of the present invention comprises contacting the NF2-deficient tumor cells or the NFl -deficient tumor cells with a HSP90 inhibitor that results in an inhibition of HSP90 function. The method further comprises contacting the NF2-deficient tumor cells or said NFl -deficient tumor cells with said HSP90 inhibitor that results in an upregulation of HSP70. Further the contact of the NF2-deficient tumor cells or the NFl -deficient tumor cells with the HSP90 inhibitor results in degradation of ErbB2 and/or phosphorylated ErbB2, in degradation of Akt and/or phosphorylated Akt or in degradation of Raf and/or phosphorylated Raf. More specifically, the contact of the NF2-deficient tumor cells or said NFl -deficient tumor cells with the HSP90 inhibitor results in a reduction in phosphorylation of proteins downstream of the ErbB2, Akt or Raf signaling pathway. The degradation or upregulation of the proteins or reduction in phosphorylated proteins is detected using an antibody.
[00243] The method also contacts a HSP90 inhibitor with the NFl -deficient and NF2- deficient cells where the HSP90 inhibitor comprises a biological agent or molecule, wherein the biological agent or molecule is selected from the group consisting of peptide, antibody, siRNA and antisense nucleic acid. [00244] In still a further embodiment, the method comprises contacting the NF2-deficent tumor and NFl -deficient tumors with at least one composition comprising at least one heat shock protein 90 (HSP90) inhibitor comprising radicicol or a derivative thereof, 17-AAG or a derivative thereof, a purine scaffold-based HSP90 inhibitor or a derivative thereof, a pyrazole scaffold-based HSP90 inhibitor or a derivative thereof, an imidazole scaffold-based HSP90 inhibitor or a derivative thereof, a tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitor or a derivative thereof, Novobiocin or a derivative thereof, or any combination thereof. Further, in a further embodiment, the HSP90 inhibitor comprises a HSP90 modulator compound such as a HDAC inhibitor and specifically HDAC6 inhibitor. This HSP90 modulator compound may further comprise Trichostatin A, SAHA, LAQ824, FK228,
53412 v2ΦC 78 or derivatives thereof. Further, the method comprises administering a HSP90 inhibitor comprising an inhibitor or compound that modulates or increases the level of HSPs such as HSP70 and HSP27. The HSP90 inhibitor further comprises NZ28 (NCS-134754) or a derivative thereof. The HSP90 inhibitor also comprises an inhibitor or compound that modulates (e.g. increases) the level of HSPs such as HSP70 and HSP27. The HSP90 inhibitor may also comprise NZ28 (NCS- 134754) or a derivative thereof. The method also comprises contacting the NF-2-deficient cells or NF-I -deficient cells are contacted with said compound and a compound that inhibits or reduces PAK activity. [00245] In another embodiment, the present invention includes a method for screening a test compound for treatment of NF-2 or NF-I comprising treating or contacting NF-2-deficient cells or NF-I -deficient cells with said test compound, wherein a degradation of one or more client proteins of HSP90 or a decrease in activity of signaling pathways associated with one or more client proteins of HSP90 or an increase in HSP70 is indicative of an efficacious treatment of NF-2 or NF-I . The method further comprises assessing inhibition of HSP90 function. The inhibition of HSP90 function results in an upregulation of HSP70. The one or more client proteins of HSP90 are selected from the group consisting of ErbB2, AKT, and Raf. Additionally, the signaling pathways are associated with one or more client proteins of HSP90 are ErbB2 pathway, AKT pathway or Raf pathway which contain at least one protein selected from group consisting of ErbB2, AKT, Raf, mTOR, GSK3, 4E-BP1 , Bad, FKHR, S6K, S6, Mek, and Erkl/2. Specifically, the one or more client proteins is AKT, the AKT is degraded by said test compound, resulting in reduced phosphorylation of AKT. The treatment results in reduced phosphorylation of S6, GSK3, FKHR, Mek, or Erkl/2. The reduced phosphorylation is detected using an antibody. [00246] The method of the present invention further comprises measuring NF-2-deficient cells or NF- 1 -deficient cells following treatment with the test compound, wherein a decrease in the number of NF2-deficient cells or NF-I -deficient cells following treatment with the test compound or a decrease in proliferation of NF2-deficient cells or NF-I -deficient cells following treatment with the test compound is indicative of an efficacious treatment. Additionally, the method further comprises comparing the NF2-deficient cells or NF-I- deficient cells following treatment to untreated NF2 deficient cells or NF-I -deficient cells, wherein a decrease in one or more client proteins of the HSP90 following treatment with the test compound compared to untreated NF2-deficient cells or NF-I -deficient cells is indicative of an efficacious treatment. Additionally, the method further comprising comparing the NF2-deficient cells or NF-I -deficient cells following treatment to untreated NF2 deficient
53412 v2/DC 79 cells or NF-I -deficient cells, wherein a decrease in number of NF2 deficient cells or NF-I- deficient cells following treatment with the test compound or a decrease in proliferation of NF2-deficient cells or NF-I -deficient cells following treatment with the test compound compared to untreated NF2-deficient cells or NF-I -deficient cells is indicative of an efficacious treatment. The treatment of the NF2-deficient cells or NFl -deficient cells with said test compound occurs in vitro or ex vivo. The NF2-deficient tumor cells are Nf2- deficient mouse Schwann cells and the NFl -deficient tumor cells are Nf 1 -deficient mouse Schwann cells. Also the NF2-deficient tumor cells are NF2-deficient human schwannoma cells and said NFl -deficient tumor cells are NFl-deficient human Schwann cells. [00247] The NF2-deficient tumor cells are selected from the group consisting of NF2- deficient schwannoma cell line cells, NF2-deficient meningioma cell line cells and NF2- deficient mesothelioma cell line cells. The NF2-deficient tumor cells are selected from the group consisting of HEI193 cells, SF1335 cells, BAR cells and RAV cells. The NFl-deficient tumor cells are selected from the group consisting of human MPNST cells, primary neurofibroma cells derived from NFl patients, mouse iV/7,7?53-deficient MPNST cell lines established from cisN/I;p53 mice, and NfJ-/- mouse cells, such as Schwann cells, mouse embryonic cells, and leukemia cells. More specifically, the NFl -deficient tumor cells are selected from the group consisting of ST88-14, 88-3, 90-8, and sNF96.2. The NF2-deficient tumor cells or NFl -deficient tumor cells that are contacted with said HSP90 inhibitor can occur in vivo. The NF2-deficient tumor cells or said NFl-deficient tumor cells are from a human, canine, rat or mouse. Processes for the Preparation of the Compounds
[00248] Modular synthetic processes directed to the synthesis of pochonin D and pochonin A were adapted to the synthesis of a library of resorcylic acid lactones that extend beyond the natural resorcylides. The syntheses developed utilize resin-assisted or solid phase synthesis to minimize and facilitate the isolation of intermediate and final products. First, a description of the synthetic protocols directed to the natural resorsylic acid lactones is presented followed by the synthesis of the library of compounds.
The following abbreviations are used herein. Ac Acetyl (CH3C=O)
ADP Adenosine diphosphate
AlBN Azobis(isobutyronitrile)
All AHyI
ATP Adenosine triphosphate
53412 v2 DC 80 BER Borohydride exchange resin
BBN Borabicyclononane
Bn Benzyl
Bz Benzoyl
CAN Ceric ammonium nitrate
CSA Camphorsulfonic acid δ Chemical shift (NMR) dba Dibenzylideneacetone
DBU l ,8-Diazabicyclo[5.4.0]undec-7-ene
DDQ 2,3-Dichloro-5,6-dicyano-l ,4-benzoquinone
DEAD Diethyl azodicarboxylate
DIAD Diisopropyl azodicarboxylate d.e. Diastereoisomeric excess
DET Diethyl tartrate
DHP Dihydropyran
DIBAL or Dibal-H Diisobutylaluminum hydride
DIC TV.iV'-diisopropylcarbodiimide
DMAP 4-Dimethylaminopyridine
DMDO Dimethyldioxirane
DMF Dimethylform amide
DMPI Dess-Martin periodinane
DMSO Dimethylsulfoxide
DNA Desoxyribo nucleic acid dppe 1 ,2-Bis(diphenylphosphino)ethane
EC5Q Plasma concentration required for obtaining 50% of maximum effect in vivo
EDC l -Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
EDTA Ethylenediaminetetraacetic acid e.e. Enantiomeric excess
EOM Ethoxymethyl (CHC3H2OCH2-)
FDA Food and Drug Administration
Fm oc 9-Fluorenylmethoxycarbonyl
GI50 Concentration required for 50% inhibition of cell growth
53412 v2/DC 8 1 Grubbs" II Grubbs" second generation catalyst: (ruthenium[l ,3-bis(2,4,6- trimethylphenyl)-2-imidazolinylidene)dichloro(phenylmethylene)
(tricyclohexylphosphane) Grubbs' II
Figure imgf000083_0001
HFIP Hexafluoroisopropanol
HMDS Hexamethyldisilazide
HMPA Hexamethylphosphorictri amide
HOBT N-Hydroxybenzotriazole
HPLC High perfomiance chromatography
HRMS High resolution mass spectrometry
HSP90 Heat shock protein 90
Hunig's Base Diisopropylethylamine
IC50 Concentration of a drug that is required for 50% inhibition in vitro imid. Imidazole
IpC2BH Bis-isopinocamphorylborane
J Coupling constant
KHMDS Potassium hexamethyldisilylamide
L.C. Liquid chromatography
LDA Lithium diisopropylamide
LiHMDS Lithium hexamethyldisilazide (LiN(SiMe3^) μM Micromolar concentration (μmol.l"1)
MAP Mitogen-activated protein mCPBA meto-Chloroperoxybenzoic acid
MOM Methoxymethyl (CH3OCH2-) mPvNA Messenger ribonucleic acid
M.S. Mass spectrum
NaHMDS Sodium hexamethyldisilazide
NMR Nuclear magnetic resonance
NMM iV-Methylmorpholine
NMO JV-Methylmorpholine-iV-oxide
NOE(SY) Nuclear overhauser effect
53412 \2'DC 82 PCC Pyridinium chlorochromate
PDC Pyridinium dichromate
PG Protecting Group
PMB /?αra-Methoxybenzyl
PNA Peptide nucleic acid
Piv Pivaloyl
PS- Polymer supported
PS-TBD ( 1 ,5,7)-Triaza-bicyclo[4.4.0]dodeca-5-ene-7-methyl polystyrene
Pyr or Py Pyridine rac Racemic
RAL Resorcylic acid lactone
RCM Ring-closing metathesis
RedAl Sodium bis(methoxyethoxy) aluminum hydride
Rr Retention factor
RNA Ribonucleic acid
RT Room temperature
SAE Sharpless asymmetric epoxidation
SAR Structure-activity relationship
SEM 2-Trimethylsilylethoxymethoxy
TBAF Tetra-n -butyl ammonium fluoride
TBAI Tetra-n-butylammonium iodide
TBDPS r-Butyldiphenylsilyl
TBHP r-Butylhydroperoxide
TBS /-Butyldimethylsilyl
Teoc 2-(Trimethylsilyl)ethoxycarbonyl
Tf Triflate (CF3SO3)
TFA Trifluoroacetic acid
TFAA Trifluoro acetic acetic anhydride
THF Tetrahydrofuran
THP Tetrahydropyran
TLC Thin layer chromatography
TMS Trimethylsilyl
Ts Tosyl (P-CH3C6H4SO2) p-TSOH /?αrα-Toluenesulfonic acid
53412 v2 DC I. Synthesis of Pochonin D
Preliminary Studies
[00249] Retrosynthetic disconnections for pochonin D (2-85) depicting the synthetic strategy are shown below. A Mitsunobu esterification, an acylation and a ring-closing metathesis are shown as the main disconnections using three building blocks: acid 2-87, alcohol (5)-2-27 and Weinreb amide 2-88.
Mitsunobu Estenf/cation
Figure imgf000085_0001
Retrosynthetic analysis for pochonin D based on radicicol and pochonin C syntheses
[00250] The Weinreb amide moiety 2-88 was synthesized as shown in Scheme 1. Thus, alkylation of intermediate 2-7 with 5-iodo-l-pentene yielded Weinreb amide 2-88 in two steps from thiophenol.
Thiophenol
Figure imgf000085_0002
Best conditions : X = I, LDA 30min at -78 °C, HMPA 30 min at -78 °C, o.n. R.T. Scheme 1 : First generation of Weinreb amide synthesis a) thiophenol (1.0 equiv.), K2CO, (1 ,0 equiv.), DMF, 23 °C: 2-chloro-iV-methoxy-iV-methylacetamide (1.0 equiv.), 23 0C. 4 h, 95 %: b) LDA (2.0 equiv.). HMPA (2.0 equiv.), 5-iodo-l-pentene (2.0 equiv.), -78 → 23 0C, 13 h, 30 %.
[00251] In parallel, an alternative synthetic pathway was developed, starting with commercially available m-6-nonen-l-ol (Scheme T),
53412 v2< DC 84
Figure imgf000086_0001
Scheme 2: Second generation of Weinreb amide synthesis a) PDC (2.0 equiv.). DMF, 23 0C, 12 h. quant; b) /Pr2NH (2.6 equiv.). πBuLi (2.2 equiv.), HMPA, CCl4 (5.0 equiv.), THF. -78 → 0 °C, 3 h, c) /V,0-diniethylhydroxylamine hydrochloride (2.0 equiv.), DMAP (cat.), EDC (2.0 equiv.), CH2Cl2, 23 0C, 4 h, 88 % (2 steps); d) /Pr2NEt (0.9 equiv.), thiophenol (0.9 equiv.), DMF, 80 0C. 12 h, 84 %; e) H2O2 (2.0 equiv.). HFIP, 23 0C. 3 h; f) toluene, 80 0C, 8 h. 75 % over 2 steps.
[00252] Following a classical oxidation procedure (PDC in DMF), acid 2-89 was then α- chlorinated by formation of the enolate using LDA and subsequent chlorine addition using carbon tetrachloride (Snider, B. B. & Kulkami, Y. S., J Og Chem 1987, 52, 307-310). After work-up, compound 2-90 was obtained as a black oil although as a pure compound by 1H NMR. Efforts to purify this acid proved disappointed and it was used directly in the following step. Further amide formation using N,(2-dimethylhydroxylamine and EDC and displacement of the chlorine atom with thiophenol afforded compound 2-92 in 74 % overall yield from cis- 6-nonen-l-ol. Via the oxidation/elimination reaction, the thioether Weinreb amide 2-92 could be converted in its closely related derivative 2-93.
[00253] Due to the high cost of commercially available 2,4-dihydroxy-6-methylbenzoic acid, a protocol was developed that allows the synthesis of 2,4-dihydroxy-6-methylbenzaldehyde which can further be derivatized in the corresponding acid using various protecting groups (Scheme 3). Starting from orcinol and following a Vilsmeier-Haack procedure, aldehyde 2- 94a was obtained in 45 % yield (72 % based on recovered S. M.). As described in the experimental section, this aldehyde precipitates at pH = 7 and is recovered with good purity (> 95 % as judged by 1H NMR).
Figure imgf000086_0002
Orcinol 2-94a-k
Figure imgf000086_0003
PG = H (a, b), Me (c, d), SEM (h), EOM (e, f, g), TBDPS (i, j, k) X = H1 CI
Scheme 3: Resorcylic acid synthesis from orcinol
53412 v2'DC 85 a) POCl., (4.0 equiv.), DMF, 0 → 23 → 80 0C, 3 h, 45 %; b) General procedure: PGCl, /Pr2NEt, CH2Cl2, 23 0C, 1 h, see experimental sections for each PG ; c) See the following table.
[00254] Compound 2-94a was then protected with different groups to generate aldehydes 2- 94c-k which were subsequently oxidized to afford the corresponding acids 2-95a-k in good yields (Scheme 3, Table 2). By varying the oxidation conditions, it was found possible to chlorinate the ring in a one pot sequence (oxidation/chlorination) in the required position for the synthesis of pochonin D.
Table 2: Oxidation conditions to yield various protected resorcylic acid
Solvent
2-95 PG, PG-, X Acid NaClO, Time5 Yield [ sytema a H H H NH2SO3H 3.25 equiv. A 1 h 86
NH2SO3H 2.0 equiv. B b H H Cl 12 h 90
C Me Me H NH2SO3H 3.25 equiv. A I h 82 d Me Me Cl NH2SO3H 3.25 equiv. B 12 h 89 e EOM EOM H NaH2PO4 5.0 equiv. C 12 h 68 f EOM EOM Cl NaH2PO4 5.0 equiv. B 12 h 89 gd EOM H H NaH2PO4 5.0 equiv. C 12 h 87 h SEM H Cl NH2SO3H 3.25 equiv. B 12 h 70 i TBDPS H Cl NH2SO3H 3.25 equiv. B 30 min 93 j TBDPS TBDPS H NH2SO3H 3.25 equiv. A 30 min 92 k TBDPS TBDPS Cl NH2SO3H 3.25 equiv. B 30 min 95 3 A: H2O/THF/DMSO (20: 10: 1) or B: H2CvTHF (2:1) or C: DMSO, b When run for more than 1 h, the reaction was heated up slowly to room temperature, c In this case, only 2 equiv. of acid were used to avoid over- chlorination, ' This compound was previously assigned as compound 2-66.
[00255] Depending on the protecting group, two different acidic buffers (NH2SO3H or NaH2PO4) and various solvent systems were used (Table 2). To avoid any chlorination reaction, a small percentage of DMSO as a mixture in THF/H2O along with sulfamic acid ((a) Lindgren, B. O. & Nilsson, Acta Chem. Scand. 27, 888-890 (1973), (b) Colombo, L et ai, J. Chem, Soc, Perkin Trans. 1, 136-140 (1980)). proved to be essential and very effective to quench HOCl (entries a, c, j).165b The one pot oxidation/chlorination sequence was done in absence of DMSO and required longer time to reach completion (entries b, d, f, h). Due to the acidic liability of the EOM protecting groups, NaH2PO4 was used as instead of sulfamic acid and the oxidation was carried out in pure DMSO. Importantly, trials to oxidize TBDPS
53412 v2'DC 86 protected aldehydes 2-94i-k in the presence OfNaH2PO4 failed and oxidation of the mono- EOM protected aldehyde 2-94g led to uncompleted reaction. This forced us to work with the bis-protected compound which could be selectively deprotected to afford the chlorinated analog of compound 2-95g in 77 % yield over the two steps {vide infra). In the first sequence used for the synthesis of this pochonin, we started with mono-MOM protected acid 2-96. Mitsunobu esterification under standard conditions (DIAD, PPh3) between this acid 2-96 and racemic alcohol 2-27 afforded the desired ester 2-97 which was further converted to the bis- protected ester 2-98 (Scheme 4).
Figure imgf000088_0001
Scheme 4: Synthesis of MOM protected Monocillin II (2-102) a) 2-27 (1.0 equiv.). P(mClPh).-, (2.0 equiv.). DIAD (2.0 equiv.), toluene, 23 0C, 3 h, 59 %; b) MOMCl (4.0 equiv.). EtZPr2N (4.0 equiv.). TBAI (cat.), DMF, 80 °C, 3 h, 78 %; c) LDA (2.0 equiv.). THF, -78 0C: 2-88 ( 1.0 equiv.), 10 min, 50 %: d) H2O2 (2.0 equiv.), HFIP. 23 0C, 3 h; e) toluene, 80 0C, 4 h, 68 % two steps (Path A). 48 % two steps (Path B): f) Grubbs* II (5 % mol), toluene (2 niM), reflux, 15 min, 63 % (path A, trans/cis 4: 1), quant, (path B, trans/cis 7: 1 ).
[00256] Acylation using previously optimised conditions (2 equiv. of LDA at -780C) allowed the formation of the acyclic precursor 2-99 along with some unreacted starting material. We envisioned two different sequences (oxidation/elimination followed by ring-closing metathesis (path A) or vice-versa (path B)) to yield compound 2-102. Both pathways proved similar (same overall yield) albeit with a higher selectivity in the metathesis reaction when following path B (cis/trans ratio 7: 1 vs. 4: 1 for path A). Having compound 2-102, MOM deprotection and chlorination of the aromatic ring should have allowed the formation of pochonin D (2-85, Scheme 5),
53412 v2 'DC 87
Figure imgf000089_0001
Scheme 5: Synthesis of racemic Monocillin II (2-103) a) HClconc (2.5 % in dioxane), 0 → 23 0C5 1 h, 79 %; b) SO2Cl2, Et2O or CH2Cl2, 0 0C, or Ca(OCl)2, Acetone, H2OZAcOH, O 0C.
[00257] Deprotection of the MOM groups following a well-known procedure allowed easily the generation of racemic monocillin II (2-103) but the chlorination proved much more challenging than expected. All attempts to chlorinate compound 2-103 with either SO2Cl2 or Ca(OCl)2 failed, leading to several by-products and only traces of desired compound 2-85 as determined by L.C./M.S. Trials of chlorination on macrocycle 2-102 led to disappointment and on compound 2-101 led mostly to the exclusive oxidation of the thioether moiety. Attempts on compounds 2-98 and 2-97 led to the isolation of some chlorinated compound albeit in low yield and along with chlorination of the terminal olefin.
[00258] As an alternative, starting with the chlorinated analog of acid 2-96 and following the two steps sequence developed for MOM-protected Monocillin II (Scheme 4), compound 2- 104 was obtained in good quantities (Scheme 6). Although acylation reaction using Weinreb amide 2-7 led to the isolation of compound 2-103 in 37 % yield, no reaction was observed when using Weinreb amide 2-88. We thought that the failure of this last reaction might be due to the thioether linkage present on the Weinreb amide moiety and decided to use directly its α,β -conjugated analog 2-93.
Figure imgf000089_0002
Scheme 6: Acylation trials with various Weinreb amide a) LDA (2.0 equiv.). THF, -78 0C; 2-7 or 2-88 or 2-93 (1.0 equiv.), 10 min, 37 % (2-105), 40 % (2-107).
[00259] Indeed, acylation reaction between the toluic anion of ester 2-104 and Weinreb amide 2-93 led to the isolation of acyclic compound 2-107 in 40 % yield along with some
53412 v2'DC unreacted starting material. Attempts to improve this yield were disappointed probably because of steric effects of the chlorine atom. However, the possibility of achieving the acylation reaction on the chlorinated toluic ester opened the door for the completion of pochonin D synthesis.
Total Synthesis of pochonin D
[00260] EOM protecting groups were chosen for phenolic protection. Using the chemistry described above to synthesize toluic acids, mono-EOM chlorinated acid 2-108 was synthesized in three steps from formylated orcinol 2-94a (Scheme 7). The selective deprotection of the ortho-phenol was achieved using a specific concentration of TFA in a THF/MeOH mixture (THF/TFA/MeOH 7: 1.5: 1 (vol.)) without any bis-deprotection. Further Mitsunobu esterification using standard protocol (DIAD, PPh3, toluene) and reprotection of the ort/io-phenol allowed the formation of compound 2-110 in 51 % yield from acid 2-108.
Figure imgf000090_0001
Scheme 7: First total synthesis of pochonin D a) EOMCl (4.0 equiv.). /Pr2NEt (4.0 equiv.), CH2Cl2, 23 0C, 1 h, 81 %; b) NaH2PO4 (5.0 equiv.). NaClO2 (5.0 equiv.), H,O/THF 2: 1 , 0 → 230C, 12 h, 89 %; c) THF/TFA/MeOH 7: 1.5: 1 , 23 0C, 45 min, 80 %; d) (S)-2-27 (1.0 equiv.), PPh3 (2.0 equiv.), DIAD (2.0 equiv.). toluene, 23 0C, 3 h, 72 %; e) EOMCl (2.0 equiv.), EtZPr2N (2.0 equiv.), TBAI (cat). DMF. 80 0C, 3 h, 70 %: f) LDA (2.0 equiv.). THF, -78 0C; 2-93 (1.0 equiv.), 10 min. 52 %; g) Grubbs' II (10 % mol), toluene (2 mM), 80 0C, 12 h, 94 %; h) TFA (20 %), CH2Cl2, 23 0C, 2 h, 72 %.
[00261] Deprotonation of the toluic ester 2-110 followed by addition of Weinreb amide 2-93 afforded the desired metathesis precursor 2-111 in 52 % yield along with some unreacted starting material. Treatment of triene 2-111 with the Grubbs" second generation catalyst173 at
53412 v2/DC 89 120 0C for 15 min afforded the desired cyclization product 2-112 in an excellent yield albeit as an unseparable mixture of cis/ trans olefins 1 :4. Metathesis reaction under thermodynamic control at 8O0C overnight174 shifted the equilibrium to the trans intermediate 2-112 with > 95 % selectivity (as judged by 1H NMR) and 94 % yield. It should be noted that this reaction could be performed at millimolar concentration without any detectable amount of dimerization or oligomerization. Importantly, the 10-membered ring macrocycle was not observed. Further EOM deprotection using TFA in dichloromethane allowed the first total synthesis of pochonin D which was found to have an identical NMR spectrum to the natural product.
[00262] For the puipose of diversity-oriented synthesis and as the presence of a thioether linkage was not possible having the chlorine atom on the aromatic ring, a more concise synthesis of pochonin D with polymer-supported reagents was developed ((a) Ley, S. V. & Baxendale, I. R., Nat Rev Drug Discov 1, 573-86 (2002), (b) Ley, S. V. et al, J. Chem. Soc, Perkin Trans. 1 23, 3815-4195 (2000)). The Mitsunobu reaction using directly 2,4-dihydroxy- 6-methylbenzoic acid was envisaged leading to an even more concise pathway to pochonin D. As (■S)-4-penten-2-ol ((S)-I-Il) is commercially available, improvements to the sequence leading to the aliphatic Weinreb amide 2-93 were undertaken. Indeed, although gram quantities could be obtained from the second generation synthesis (Scheme 2), several purifications were necessary. To circumvent this problem, a protocol using solid-support was developed. Commercially available 2-chloro-Λf-methoxy-yV-mefhylacetarnide (Scheme 8) was selectively S-alkylated with 3-mercaptophenol using one equivalent of base, and then loaded onto Merrifield resin in the same pot reaction by the successive addition of a second equivalent Of K2COa, the resin, and raising the temperature to 50 0C, as previously described in Chapter II (Part II).
Figure imgf000091_0001
2-chioro-W-methoxy- W-methyiacetamide b) H2O2 HFIP
O
,,0Me
N O O c) fBuOK, Me ,s. ,,0Me d) Λ
Me
2-114 2-113
Scheme 8: Solid-phase synthesis of Weinreb amide 2-114
53412 v2 'DC 90 a) 3-mercaρtoρhenol (1 ,0 equiv.), K2CO, (1.0 equiv.). DMF, 23 0C; after 8 hours, K2CO1 (1.7 equiv.), Merrifield resin, TBAI (cat.), 12 h, 50 0C. 98 %; b) H2O2 (2.0 equiv.), HFIP/CH2C12 1 : 1 , 12 h; c) fBuOK (1.0 equiv.), 5- iodo-1 -pentene (1.0 equiv.), DMSO, 23 0C, 3 h: d) toluene, 80 0C, 8 h, 77 %.
[00263] While preparations of thiol resins have been reported by direct lithiation of the resin followed by a quench with elemental sulfur, (Fan-all, M. J. & Frechet, J. M. J., J Org Chem 41, 3877-82 (1976)). this method was found more practical as it affords the polymer-bound Weinreb amide 2-49 in one step. Oxidation of the thioether 2-49 to the corresponding sulfoxide 2-113 was carried out using the aforementioned procedure involving H2O2 in HFIP/CFbCl?.163 This oxidation procedure was found practical and reliable with no over- oxidation to the sulfone and easy recycling of the fluorinated solvent as the reaction was earned out on solid phase. The amidosulfoxide 99 was then deprotonated with /1BuOK, and the resulting enolate was quenched with 5-iodo-l -pentene. The use of DMSO was found crucial for the success of this reaction, DMF, /BuOH, and 1 ,4-dioxane giving only poor results. However, the polar nature of DMSO was unfavorable for the sulfoxide elimination, and the reaction could be heated up to 60 0C without any elimination. The use of solid- support was again valuable in cleaning the compound from DMSO. Resuspension of the resin in toluene and heating up to 80 0C released, after elimination, the desired fragment 2-114 with 77 % yield and 95 % purity (judged by 1H NMR). This methodology on solid support was found very practical in terms of yield and purity of the building block 2-114 as no column chromatography was needed.
[00264] Based on the chemistry developed in solution (Scheme 7), a selective Mitsunobu esterification of 2,4-dihydroxy-6-methylbenzoic acid (2-95a) with (S)-4-penten-2-ol {{S)-2- 27) using polymer-bound DEAD afforded ester 2-116 (Scheme 9). The use Of (OiClPh)3P was found essential to suppress any competing ether formation with thepαra-phenol.182 Protection of both phenols with EOM groups afforded non-chlorinated ester 2-117, which could be used in the subsequent alkylation without further purification.
Figure imgf000092_0001
53412 v2'DC 91 Scheme 9: Synthesis of intermediates 2-110 and 2-117 using polymer-bound reagents a) NaClO2 (5.0 equiv.). NH2SO,H (5.0 equiv.), CH3CHO (1.0 equiv.), THF/H2O 5: 1 , 0 0C, 0.5 h, 92 %; b) PS- DEAD (2.5 equiv.. 1.3 mmol.g" '). (S)-4-penten-2-ol (1.0 equiv.), P(mClPh)3 (2.0 equiv.), CH2Cl2, 23 0C. 0.5 h, 68 % for 2-117 and 65 % for 2-110; c) JPr2EtN (4.0 equiv.), EOMCl (4.0 equiv.), TBAI (cat.), DMF, 80 0C, 5 h, 95 %.
[00265] In parallel and as the chlorine atom could not be introduced at a later stage (vide supra), the halogen was thus introduced prior to esterification using HClO generated in situ by the oxidation of acetaldehyde with NaClCWsulfamic acid. Acid 2-95b was obtained from its non-chlorinated parent 2-95a in 92 % yield without seeing any over-chlorination. Esterification of this product under the same conditions as for 2-95a afforded compound 2- 115. Further bis-protection with EOM groups led to ester 2-110 which could also be used in subsequent reactions without any purification. Deprotonation of the toluic esters 2-110 and 2- 117 followed by addition of Weinreb amide 2-114 afforded the desired metathesis precursors 2-118 and 2-119 which could be used directly in the following step (Scheme 10). When the acylation reaction was performed on the non-chlorinated ester 2-117, 20 % of product stemming from a 1 ,4-conjugated addition on the Weinreb amide was obtained.
Figure imgf000093_0001
X = H : 2-119 X = 01 : 2-118 b) Grubbs Il
Figure imgf000093_0002
Scheme 10: Synthesis of pochonin D (2-85) and monocillin II (2-103) using supported reagents a) LDA (2.0 equiv.). THF, -78 0C; 2-114 (1.0 equiv.), 10 min. Amberlite IRC-50 (20.0 eq. 10.0 mmol.g''): b) Grubbs" II (10 % mol), toluene (2 inM), 80 0C. 12 h. 40 % for 2-120 and 44 % for 2-112 after two steps; c) PS- TsOH ( 10 equiv.. 3.2 mmol.g'1). MeOH. 40 0C. 4 h, 90 % for 2-85 and 92 % for 2-103.
[00266] Treatment of crude trienes 2-118 and 2-119 with the Grubbs' second generation catalyst under thermodynamic control at 800C overnight led exclusively (more than 95 % selectivity) to the trans macrocycles 2-112 and 2-120. A simple filtration through a path of silica was then used to remove all of the catalyst and its by-products, affording compound 2-
53412 v2/DC 92 112 in 44 % yield over two steps. While the metathesis reaction earned out on purified triene 2-118 was nearly quantitative, it was found more practical to carry out the whole synthetic sequence from compound 2-95a without any purification, thus affording the protected pochonin D 2-112 in 25 % yield over five steps. For compound 2-120, a column chromatography was necessary to separate the 12-membered macrocycle 2-121 coming from the side reaction (1 ,4-addition) occurring during the acylation reaction, Removal of EOM groups from both macrocycles 2-112 and 2-120 using sulfonic acid resin in MeOH allowed the synthesis of both pochonin D (2-85) and monocillin II (2-103) in 90 % and 92 % yield, respectively. As shown for the acylation reaction, the presence or absence of the chlorine atom on the aromatic ring seems to influence the reactivity of the conjugated olefin. Indeed, deprotection of compound 2-120 with HCl (2.5 % in dioxane) led to the conjugated addition of the chlorine ion, whereas compound 2-112 could be deprotected with HCl to obtain pochonin D (2-85). This synthesis using polymer-supported reagents allowed the achievement of pochonin D and monocillin II in six (23 % yield) and five (24 % yield) steps respectively. Starting from commercially available building blocks, only one chromatographic purification of the final compound is required for all the synthetic pathway and this methodology could be used for the synthesis of libraries. Evaluation for HSP90 affinity in a competition assay with geldanamycin revealed that pochonin D is a good ligand for HSP90 with an IC50 of 8OnM as compared to 2OnM for radicicol (vide infra). II Synthesis of pochonin A
[00267] Having access to pochonin D (2-85), pochonin A (2-122) was then prepared not only to confirm its structure but also to compare its biological activity to pochonin D and radicicol.
Figure imgf000094_0001
2-1 2-122 2-85
Radicicol Pochonin A Pochonin D
[00268] Epoxidation of pochonin D using DMDO allowed the formation of pochonin A as a 1 : 1 mixture of diastereoisomers that could be separated by column chromatography. Attempts to improve the selectivity of this reaction using epoxone ((a) Tu, Y. et al, J Am Chem Soc 118, 9806-9807 (1996), (b) Wang, Z.-X. et al., J Org Chem 62, 2328-2329 (1997)) as a chiral auxiliary failed completely with no conversion at 0 0C or at room temperature. The lack of solubility of pochonin D at low temperature did not allow for improvement of this selectivity and other routes were pusued. As depicted in Scheme 1 1, the epoxidation of the bis-EOM
53412 v2'DC 93 protected pochonin D (2-112) did not improve the selectivity and deprotection of the EOM in the presence of the epoxide did not give satisfactory results under a variety of conditions.
Figure imgf000095_0001
2-124 2-125
Scheme 11: Direct conversion of bis-EOM pochonin D (2-112) a) DMDO (1.0 equiv.), CH3CN, O 0C → 23 °C. 1.5 h , 79 %; b) PS-TsOH (10.0 equiv., 3.2 mmol.g"'), MeOH, 40 0C, 1 h.
[00269] The suitability of silyl based protecting groups to access this type of molecules was investigated. This was also motivated by the fact that it was known that a silylated carboxylic acid could be converted to an acid chloride under acid-free conditions which are compatible with silyl protected phenols (Wisnner, A. & Grudzinskas, C. V., J Org Chem 43, 3972-3974 (1978)). Thus, persilylation of benzoic acid 81b (Scheme 12), followed by "acid-free" conversion of the silyl ester to the corresponding acyl chloride yielded key intermediate 112 upon esterification with alcohol [R)-Il . The low yield reported for this three steps sequence was attributed to the lability of the ortho-IBS group. Attempts to improve the efficiency of this protocol either by purifying the persilylated intermediate (column on neutralized silica, filtration on neutral alumina, distillation or azeotrope to evaporate the excess of TBSCl) or by isolating the acid chloride only led to disappointing results.
Figure imgf000095_0002
53412 v2/ DC 94 Scheme 12: Pochonin A synthesis using TBS-protecting groups a) JPr2EtN (6.0 equiv.), TBSCl (3.0 equiv.), CH2Cl2. 23 0C, 3 h; b) Oxalyl chloride (1.0 equiv.). DMF (cat.), CH2Cl2, 0 → 23 0C, 1 h; c) Et3N (2.26 equiv.), Λ-(-)-penten-2-ol (3.0 equiv.), DMAP, 0 -→ 23 0C, 12 h, 29 % over 3 steps; d) LDA (2.0 equiv,), THF, -78 0C, Weinreib amide 100 (1.0 equiv.), 10 min, 35 %; e) Grubbs' II (10 % mol), toluene (2 tnM). 80 0C, 12 h, 79 %; f) CF3COCH3, NaHCO3 (7.0 equiv.), Oxone (4.7 equiv.), Na2 «EDTA (4 x 10"4M), CH3CN/Dimethoxymethane, 0 0C, 2 h, 83 %; g) TBAF (2.2 equiv.), THF, 23 0C, 20 min, 80 %, 3: 1 mixture of diastereoisomers.
[00270] Deprotonation of the toluic ester 2-126 followed by reaction with Weinreb amide 2- 114 afforded metathesis precursor 2-127 in modest yield. Ring-closing metathesis using Grubbs' second generation catalyst17"' under aforementioned thermodynamic conditions174 (80 0C, overnight) afforded macrocycle 2-128 in good yield and excellent cisl trans ratio (< 5 % cis). Epoxidation of the non-conjugated olefin was optimal when carried out at 0 0C with methyl(trifluoromethyl)-dioxirane generated in situ (Yang, D., et al. J. Org. Chem,,, 60, 3887-3889 (1995) affording TBS-protected pochonin A (2-129) in excellent yield as an inseparable 3: 1 diastereoisomeric mixture. Attempts to further improve the stereoselectivity of the epoxidation by reducing the temperature (-10 0C) or using epoxone were not productive. Deprotection of compound 2-129 using classical silyl deprotection conditions (TBAF in THF) afforded a separable diastereomeric mixture and confirmed that the major product was indeed the desired pochonin A (2-122). ' [00271] Alternative protecting groups were evaluated to improve the yield of the esterification and acylation reactions. Based on their stability toward basic conditions but also on their liability towards TBAF, SEM protecting groups were considered. Following the procedure described for the polymer-assisted synthesis of pochonin D, selective Mitsunobu reaction between benzoic acid 2-95b and chiral alcohol (S)-2-27 using polymer-bound DEAD and subsequent protection with SEM-Cl afforded ester 2-130 in 72 % yield (Scheme 13).
53412 v2'DC 95
Figure imgf000097_0001
Scheme 13: Pochonin A synthesis using SEM-protecting groups a) PS-DEAD (2.5 equiv., 1.3 mmol.g"1). 5-(-)-4-penten-2-ol (2.0 equiv.), P(OTClPh)1 (2.0 equiv.), toluene, 23 0C, 10 mm; b) NaH 60 % (4.0 equiv.), SEMCl (4.0 equiv.), THF, 0 0C, 2 h, 72 % over 2 steps; c) LDA (2.0 equiv.), THF. -78 0C. Weinreib amide 2-114 (1.0 equiv.), 10 min, 60 %; d) Grubbs' II (10 % mol), toluene (2 mM), 80 0C. 12 h, 87 %; e) DMDO (1.0 equiv.), CH3CN, 0 → 23 0C, 1.5 h, 83 %, 1 : 1 mixture of diastereoisomers; f) MgBr2-Et2O (8.0 equiv.), CH2Cl2, 23 0C, 1 h. 70 %.
[00272] Acylation of toluate ester 2-130 using Weinreb amide 2-114 led to the isolation of the acyclic precursor 2-131 in 60 % yield. Treatment of the triene 2-131 with the Grubbs' second generation catalyst under thermodynamic conditions (80 0C, overnight) afforded the corresponding macrocycle 2-132 in 87 % yield (< 5 % cis olefin), which was epoxidized under the same conditions as for the TBS-protected compound 2-128 [methyl(trifluorornethyl)-dioxirane], yielding compound 2-133 in 83 % yield albeit in a 1 :1 diastereomeric ratio (inseparable). All attempts to deprotect the SEM groups using TBAF were unsuccessful (even when performed on macrocycle 2-132), leading mostly to the formation of isocoumarin. Other methodologies were then examined such as sulfonic acid resin in MeOH at 40 0C, Montmorillonite Kl O in toluene at 50 0C and MgBr2^Et2O in dichloromethane at room temperature. The use of either sulfonic acid resin or Montmorillonite Kl O led to the opening of the epoxide. However, 8.0 equiv. of MgBr2^Et2O afforded the desired pochonin A (2-122) along with its diastereoisomer (2-122b) as a separable mixture. The yield was limited by reaction conversion, since longer times led to the formation of the corresponding bromohydrine. In the case of EOM-protected product 2-123, treatment with MgBr2 was found to open the epoxide faster than to deprotect the EOM. Pochonin A was found to be a good ligand of HSP90 with an IC50 of 9OnM (vide infra).
53412 v2 DC 96 III. Diversity Oriented Syntheis of Pochonin Analogues
[00273] With the objective to extend the diversity of the RAL, we turned our attention to the synthesis of a library. This library was envisioned to stem from five points of diversity around the resorcylic macrolide scaffold: modifications of the /?αra-phenol (R1, a number of natural resorcylides bearing a methyl group at this position), the group on Cl 7 (R2, both stereochemistry are present in natural resorcylides; however, only with a methyl substituent),
Mitsunobu estenfication
Figure imgf000098_0001
General structure and retrosynthetic analysis of the library
the C14-C15 olefin (R3), the C9 carbonyl (R4), the olefin ClO-Cl 1, and the meta position on the aryl ring (R" , a number of natural resorcylides bear a chlorine at this position). To minimize traditional chromatography, a prerequisite in designing the chemistry used to elaborate the molecular diversity of this scaffold was its ability to be earned out by using polymer-bound reagents. Therefore, the assembly of the macrocycle was thought to rely on the chemistry developed for the synthesis of pochonin D using polymer-bound reagents (Schemes 9 and 10).
[00274] A variety of homoallylic alcohols 2-134 bearing various substituent at R2 was envisioned. Homoallylic alcohols 2-134 that are not commercially available may be obtained in by any suitable method. In one embodiment, the homoallylic alcohols 2-134 were obtained in their highest enantiomeric form either by enzymatic resolution2 of the racemic alcohol or by means of Brown allylation168 of the corresponding aldehyde (H. E.Master et αl., Tet. Lett,, 37:9253 (1996); S. Singh et αl., Tet. Asymm., 13:2679 (2002) or via Brown allylation of the corresponding aldehyde. (H. C. Brown and P. K. Jadhav J. Am. Chem. Soc, 105:2092 (1983). The phenyl (2-134a), the pyridinyl (2-134b) and the furyl (2-134c) alcohols were prepared by enzymatic resolution (Scheme 14). Racemic alcohols 2-134a-c were obtained after Grignard addition of commercially available allylmagnesium bromide on their corresponding aldehyde 2-134a-c.
53412 v2, DC 97
Figure imgf000099_0001
(R)-2-137a-c (R)-2-134a-c e.e. > 89%
Scheme 14: Synthesis of chiral alcohols 2-134a-c using enzymatic resolution a) AlIyIMgBr (1.5 equiv.). THF, 0.5 h, 0 0C, 71 % (2-134a), 41 % (2-134b), 74 % (2-134c); b) R2=Ph: vinyl acetate (32.5 equiv.), Amano Lipase PS-C II (50mg/mmol of 2-134). 23 0C, 30 h (monitored by 1H NMR), R2=Pyr. Fur: vinyl acetate (10.0 equiv.), Amano Lipase PS-C II (50mg/mmol of 2-134), THF, 23 0C, 5-30 h (monitored by 1H NMR): c) K2CO3 (0.8 equiv.), MeOH, 23 0C, 98 % ((R)- 2-134a), 92 % ((R)- 2-134b), 84 % ((R)- 2-134c).
[00275] Kinetic enzymatic resolution of racemic alcohols 2-134a-c was realized using the highly efficient Amano lipase (an immobilized version of Pseudomonas cepacia). This enzyme catalyzed a selective transesterification of alcohols (7?)-2-134a-c with vinyl acetate as an acyl donor, the (S) alcohols 2-134a-c being isolated in excellent yields and good enantiomeric excesses (Table 3).
Table 3: Enantioselective acylation of alcohols rαc-2-134a-c by transesterification with lipase
Conv. Entry Substrate Time (h) Ratio Yield ( %) e.e. ( %) Yield ( %) e.e. ( %)
( %)
(OH/OAc) (S)- 2-134 (5)-2-134 (i?)-2-134 (Λ)-2-134
1 rac-2-134a 30 50:50 45 98 49 93
2 rac-2-134b 30 52:48 50 89 39 94 rαc-2-134c 5 49:51 44 88 49 89
[00276] Enantiomeric excess obtained with this methodology are all above 88 % and crucially depends on the conversion of the reaction. Acetylated alcohols (i?)-2-137 were then hydrolysed to the corresponding alcohols (7?)-2-134a-c in excellent yields. In addition, a second process based on Brown allylation was developed for the synthesis of the isopropyl (2- 134d), the propyl (2-134e) and the benzyl (2-134f) alcohols (Scheme 15).
53412 v2/DC 9g 80% (iPr) (-)-α-pin 91% (Pr)
Figure imgf000100_0001
95% (Bz)
Scheme 15: Synthesis of chiral alcohols 120d-f using Brown allylation a) (-)-α-pinene (2.4 equiv.), BH3-Me2S (1.0 equiv.), THF, 23 0C for 1 h and then 4 0C for 12 h, 76 %; b) MeOH ( 1.2 equiv.), Et2O. 0 0C. 2 h, 94 %; c) AlIyIMgBr (0.95 equiv.), Et2O, 0 → 23 0C, 1 h, 92 %; d) 122d-f (1.05 equiv.), Et2O, -100 0C. 0.5 h; 3N NaOH, H2O2 35 %, reflux. 3 h, 77-93 %. Enantiomeric excesses of alcohols were determined by chiral HPLC analysis after acylation with 3,5-dinitrobenzoyl chloride.
[00277] (-)-B-Allyldiisopinocampheylborane (2-139, (-)-Ipc2BAllyl) was synthesized in a three steps sequence from (-)-α-pinene involving an hydroboration, the formation of the corresponding MeO-borinic ester 2-138 and its treatment with a Grignard reagent. Further condensation on aldehydes 2-134d-f followed by oxidation of the resulting borinates with alkaline hydrogen peroxide allowed the formation of the chiral homoallylic alcohols 2-134d-f in good enantiomeric excess.
[00278] The macrocycle assembly was modeled after the synthesis of pochonin D. (E. Moulin, V. Zoete, S. Barluenga, M. Karplus, N. Winssinger, J. Am. Chem. Soc, 127:6999 (2005)). Thus as shown in Scheme 16, commercially available benzoic acid 2-95a and its chlorinated analog 2-95b (the chlorine atom was introduced on acid 2-95a prior to esterification using HClO generated in situ by the oxidation of acetaldehyde with NaClO2 /sulfamic acid were esterified with fifteen different homoallylic alcohols using polymer- supported DEAD to yield esters 2-115a-g and 2-116a-g in excellent purity. (E. Moulin et al, J. Am, Chem. Soc, 127:6999 (2005)). The products 2-115a-g and 2-116a-g were then protected with ethoxymethylene chloride (EOM-Cl) in the presence of Hunig's base to obtain the corresponding protected toluic esters 2-110a-g and 2-117a-g which could be used in the subsequent carbon-acylation reaction without further purification.
53412 v2'DC 99
Figure imgf000101_0001
Scheme 16: Synthesis of macrocylic precursors 2-112a-g, 2-120a-g and 2-121a-g a) NaClO2 (5.0 equiv.). NH2SO1H (5.0 equiv.), CH,CH0 (1.0 eqmv.). THF/H2O 5: 1 , O 0C, 0.5 h, 92 %; b) PS- DEAD (2.5 equiv.. 1.3 mmol.g"'). (R)- 2-134a-g or (S)- 2-134a-g ( 1.0 equiv.). P(mClPh), (2.0 equiv.), CH2Cl2. 23 0C, 0.5 h. 60-80 %; c) 1Pr2EtN (4.0 equiv.), EOMCl (4.0 equiv.), TBAI (cat.), DMF, 80 0C. 5 h, 80-90 %; d) LDA (2.0 equiv,). THF. -78 0C. 2-114 ( 1.0 equiv.), 10 min. Amberlite IRC-50 (20.0 eq. 10.0 mmol.g"1); e) Grubbs' II (10 % mol). toluene (2 mM). 80 0C, 12 h. 38-70 % after two steps.
[00279] Deprotonation of the toluic esters 2-110a-g and 2-117a-g using two equivalents of LDA, followed by addition of the α,β-unsaturated Weinreb amide prepared via solid phase chemistry (E. Moulin et ai, J. Am. Chem. Soc, 127:6999 (2005)) afforded acylation products 2-118a-g and 2-119a-g. The reaction was quenched with a polymer bound acid which also sequestered all the diisopropyl amine. This reaction can lead to some level of 1 ,4-conjugate addition product. (S. Barluenga et al, Chem. Eur. J, 1 1 :4935 (2005)). While the bulky chlorine present in pochonin D suppresses this reaction, compounds lacking the aryl chloride afforded 20% of the conjugate addition products 2-140a-g. Nevertheless, the crude mixtures
53412 v2 'DC 100 of these reactions were used in the subsequent cyclization step. The trienes were then subjected to ring closing metathesis using Grubbs' second generation catalyst (A. K. Chatterjee et al, J. Am. Chem. Soc., 122:3783 (2000); M. Scholl et al, Org. Lett., 1 :953 (1999)) under thermodynamic conditions (C. W. Lee, and R. H. Grubbs, Org. Lett., 2:2145 (2000)), affording the desired 14-membered macrocycles. In the cases where metathesis reactions were earned out with a mixture of 2-118a-g, 2-119a-g and 2-140a-g, the corresponding 12-membered ring product 2-121a-g was obtained in addition to 2-112a-g and 2-120a-g as a separable mixture. All successful reaction sequences were purified by standard chromatography at this stage yielding the macrocycle 2-112a-g and 2-120a-g and 2-121a-g in 30-60% and 8- 10% overall yield respectively from 2-95.
[00280] Macrocycles 2-112a-g and 2-120a-g were then used as the starting point for further diversifications. Deprotection of the EOM groups of 2-112a-g and 2-120a-g using sulfonic acid resin afforded compounds 2-103a-g and 2-85a-g in pure form and excellent yields after simple filtration of the resin and evaporation of the solvents (Scheme 17). The 12-membered ring products 2-121a-g were deprotected just as smoothly (not shown). As expected, EOM deprotection under acid catalysis were slower for the chlorinated analogues and had to be monitored as prolonging the reaction times could lead to conjugate additions. Treatment of 2- 112a-g and 2-120a-g with reducing agents led to either carbonyl reduction using Dibal or mixtures of carbonyl and 1 ,4-reduction with NaBH4. It is known that using non- C(O)ORdinating counter ion for borohydride can favor the carbonyl reduction (H. W. Gibson and F. C. Baily, J. Chem. Soc. Chem. Commun., 1977:815; A. Kirschning, J. Prakt. Chem., 2000:342). This was most conveniently achieved using a polymer supported quaternary ammonium borohydride known as borohydride exchange resin (BER). Thus, ketones 2-112a- g and 2-120a-g could be reduced using BER-resin to obtain both diastereoisomers of 2-14Ia- g in -60% yield. Deprotection of the EOMs with sulfonic acid resin under regular conditions afforded compounds 2-142a-g. Acetylation of the reduced intermediates 2-141a-g using PS- NMM/ Ac2O yielded compounds 2-143a-g which led to elimination upon deprotection to afford trienes 2-144 as a mixture of olefin geometries.
53412 v2,'DC 101 S
Figure imgf000103_0001
Scheme 17: Deprotection and Synthesis of the Reduced Ketone Analogues. a) PS-TsOH (10.0 equiv.. 3.2 mmol.g"1), MeOH, 40 0C, 4 h, > 90 %: b) BER resin ( 1.0 equiv., 2.5 mmol.g"'), MeOH, O 0C, 12 h. -60 %; c) Ac2O (1.2 equiv.), PS-NMM (1.2 equiv.. 3.20 mmol.g"1), DMAP (0.05 equiv.), DMF, 23 0C. 0.5 h, -80 %. BER resin = borohydride exchange resin, PS-TsOH = sulfonic acid resin MP, DIBAL = diisobutylaluminum hydride. DMAP = dimethylaminopyridine, DMF = dimethylformamide, PS-NMM = morpholinomethyl polystyrene.
[00281] Prolonged exposure of resorcylides 2-112a-g and 2-120a-g to methanol in the presence of sulfonic acid resin was found to lead to conjugate addition; this observation was exploited to drive the reaction to completion cleanly. Thus phenol 2-85a-g was quantitatively converted to product 2-145a-g in 15 h (Scheme 18). This product could obviously be obtained directly from 2-120a-g under the same conditions.
Figure imgf000103_0002
Scheme 18: Synthesis of Analogues 2-145 by Conjugate Addition, a) PS-TsOH (10.0 equiv., 3.2 mmol.g"1), MeOH. 40 0C. 15 h, 80 %.
[00282] Compounds 2-lO3a-g and 2-85a-g were also used as the starting points for further diversifications (Scheme 19). Thus, treatment of 2-103a-g and 2-85a-g with polymer-bound cyanoborohydride afforded the 1 ,4-reduction products 2-146 in moderate yields. The more
53412 v2 DC 102 acidic /?αrø-hydroxyl groups of 2-103a-g and 2-85a-g were substituted via either Mitsunobu reaction using polymer bound DEAD or alkylation using a polymer-bound base to afford compounds with general structure 2-147 and 2-148 respectively. Oxidation with OsO4 afforded the dihydroxylation products 2-149 as a mixture of isomers as well as the products corresponding to the dihydroxylation of the conjugate olefin (product not shown). Treatment of 2-103a-g and 2-85a-g with freshly prepared dimethyldioxirane led to the selective epoxidation of the non-conjugated olefin as a mixture of distereoisomers of pochonin A analogues 2-150. Although higher diastereoselectivity may be obtained for pochonin A if the phenols are protected with TBS (E. Moulin et ai, Org. Lett,, 7:5637 (2005)), protection was not needed here.
Figure imgf000104_0001
2-147 2-148 2-149
R2 = AIIyI, Me
R3X = EOMCI, BrCH2COOfBu
Scheme 19: Derivatization of Compounds 2-85a-g and 2-103a-g.
a) PS-TMABH1CN (2.0 equiv.. 3.5 mmol.g'1), CH2Cl2/Ac0H 10 : l, 23 0C, 4 h, -50 %; b) PPh, (2.0 equiv.), R2OH (2.0 equiv.), PS-DEAD (2,0 equiv., 1.3 mmol.g"1), CH2Cl2, 23 0C, 8 h, -60 %; c) R3X (0.9 equiv.), PS- TBD (2.0 equiv.. 2.9 mmol.g"1), CH2Cl2. 23 0C. 3 h, -90 %; d) OsO4 (0.1 equiv.), NMO (1.0 equiv.), Acetone/H2O 10 : 1, 23 °C, 1 h, > 70 %; e) DMDO (1.2 equiv., 0.04 M in acetone), CH3CN, 0 0C, 30 min, > 90 %. AIlOH = Allylalcohol, DMDO = dimethyldioxirane, NMO = 4-methylmorρholine iV-oxide, PS-DEAD = ethoxycarbonylazocarboxymethyl polystyrene, PS-TBD = TBD-methyl polystyrene, PS-TMABH3CN = (ρolystirylmethyl)trimethylammonium cyanoborohydride.
[00283] It is interesting to note that the conjugated olefin proved to have different reactivity depending on the presence or absence of the chlorine atom on the aryl ring. Whereas EOM deprotection of compound 2-120 where X - Cl and R = Me could be earned out with HCl in
53412 v2/ DC 103 dioxane, treatment of the corresponding compound 2-112 where X = H and R = Me by the same conditions led to the conjugate addition of the chlorine ion during the deprotection, affording compound 2-151 (Scheme 20). The difference in reactivity may be attributed to the different conformation of this compound. It is conceivable that the differences in conformation affect the level of conjugation between the olefin and the adjacent carbonyl group. Nevertheless, the β-chlorine could be cleanly eliminated in the presence of polymer- bound base to recover the conjugate compound 2-103a-g.
Figure imgf000105_0001
a) HCI/dioxane
Figure imgf000105_0002
, OCH2COOH, OAIIyI, OTHP,
Figure imgf000105_0003
Scheme 20: Derivatization of Macrocycles 2-120 and 2-120a-g.
a) HCl (2.5 % in dioxane), 23 0C, 3 h, > 75 %: b) PS-TBD (0.5 equiv., 2.6 mmol.g"'), CH2Cl2, 23 0C, 8 h. -90 %: c) PS-TsOH (1.0 equiv., 3.2 mmol.g 1), DHP (1.0 equiv.), CH2Cl2, 23 0C, 5 h, -80 %: d) R2ONH2.HC1 (5.0 equiv.), Pyr/AcOH 5: 1 , 40 0C, 12 h, -90 %; e) PS-TsOH (10.0 equiv., 3.2 mmol.g"1), MeOH, 40 0C, 4 h, -80 % f) PS-TsOH (cat., 3.2 mmol.g"1), DHP (1.0 equiv.), CH2Cl2. 23 0C. 5 h, -70 %; g) TFA (20 %), CH2Cl2, 23 0C, 2 h. DHP = dihydropyran, PS-TBD = TBD-methyl polystyrene, PS-TsOH = sulfonic acid resin.
[00284] While evaluating protecting groups for the phenols, it was noticed that dihydropyran, in the presence of a strong acid such as sulfonic acid, led to electrophilic aromatic substitution rather than phenol protection, (see also T. Kometani et al, Synthesis, 1988: 1005). Applying
534 ! 2 v2 DC 104 these conditions to compounds 2-103 (Scheme 20) afforded 2-153 as a separable mixture of diastereoisomers.
[00285] The formation of oxime proved to be sluggish for compounds with unprotected phenols. However, compounds 2-120 (X = H, Scheme 20) protected with EOM groups underwent smooth oxime formation with nine different hydroxyl amines to obtain compounds 2-154 as E/Z mixtures with variable ratios. EOM deprotection of 2-154 with sulfonic acid resin in methanol followed by treatment with sulfonic acid resin in DCM in the presence of dihydropyran afforded oximes 2-155 bearing a pyran substitution on the aromatic ring as a mixture of diastereoisomers. In the case where the side chain contains an acid (R2X = OCH2COOH), the deprotection of the EOM with the sulfonic acid resin in methanol was accompanied by esterification of the carboxylate. Treatment of compounds 2-12Oa and 2- 112a with trifluoroacetic led to the formation of trifluoroacetate 2-152. Finally, all attempts at oxime formation in the chlorinated analogues with or without EOM protecting groups generated mostly the corresponding 1 ,4-addition of the hydroxylamines (Scheme 21). Surprisingly, when pochonin D was protected with TBS groups, (2-128a-g, Scheme 21) the formation of the desired oxime 2-157a-g was the only product observed under the same reaction conditions. Deprotection of the TBS groups was than achieved using TBAF to obtain oximes 2-158a-g. Treatement of pochonin D with amines RONH2 led to the formation of the 1 ,4-addition product 2-156. This difference in reactivity is presumably due to the level of conjugation between the olefin and the carbonyl, which in turn is a product of the conformation of the macrocycle. It is suspected that the bulky protecting group on the ortho phenol has a steric effect on the macrocycle conformation, as compared to the phenol which forms a hydrogen bond to the carbonyl. Such effect are known to affect the efficiency of ring closing metathesis reactions (E. A. Couladouros et al., Org. Lett,, 6:977 (2004)) and epoxidation diastereoselectivity. (E. Moulin et al, Org. Lett,, 7:5637 (2005)).
53412 v2, DC 105
Figure imgf000107_0001
Scheme 21 : Oxime Formation with Compounds 2-85. a) TBSCl (5.0 equiv.), imid. (5.0 equiv.). DMF. 23 0C, 3 h, -90 %: b) RONHkHCl (5.0 equiv.), Pyr/AcOH 5: 1 40 0C, 12 h, -90 %; c) TBAF (2.5 equiv.), THF, 23 0C, 2 h, -80 %. DMF = dimethylformamide, Imid. = imidazole. TBAF = tetrabutylammonium fluoride, TBS-Cl = tert-butyldimethylsilyl chloride.
[00286] In another embodiment, the bis-methylated compounds 2-164 were prepared (Scheme 22). Acid 2-108 was used in a standard Mitsunobu esterification with alcohol 2- 159. Compound 2-160 was protected as the ort/zø-phenol followed by acylation reaction with Weinreb amide 2-114 to yield the acyclic precursor 2-162. Ring-closing metathesis followed by removal of the EOM protecting groups on compound 2-163 using sulfonic acid resin furnished the bis-methylated analog 2-164.
Figure imgf000107_0002
Scheme 22: Synthesis of bis-methyl substituted analog (2-164) of pochonin D a) VinylMgBr (2.0 equiv.). CuI (0.3 equiv.), Et2O, -30 → 23 0C, 12 h, 65 %; b) 2-159 ( 1.0 equiv.). PPh1 (2.0 equiv.), DIAD (2.0 equiv,). toluene, 23 0C, 3 h, 23 %; c) EOMCl (2.0 equiv.), NaH 60 % (2.0 equiv.), THF, 0
53412 v2 DC 106 0C, 2 h. 66 %, d) LDA (2 0 equiv ), THF. -78 0C, 2-114 (1 0 equiv ), 10 mm, 57 %: e) Grubbs" II (10 % mol), toluene (2 mM), 80 0C, 12 h, 57 %, f) PS-TsOH (10 equiv., 3.2 mmol.g"1), MeOH, 40 0C, 2 5 h, 40 %.
[00287] Additionally, oxime derivatives 2-165 and 2-167 were synthesized from macrocycle 2-163 as a separable mixture with the 1 ,4-addition product (Scheme 23). The carboxylic acid moiety of oxime 2-167 was then esterified to form the corresponding piperidine amide oxime 2-168.94 Removal of the EOM groups using sulfonic acid resin allowed the isolation of both oximes 2-166 and 2-167 from 2-165 and 2-168 respectively. These two compounds along with their parent compound 2-164 and the 1 13 compounds from the library (Figure 39) were then tested in biological assays for treating NFl and NF2.
EOMO O EOMO O EOMO O
O a) NH2OCH2COOH c) Piperidine O
Figure imgf000108_0001
Scheme 23: Synthesis of oxime derivatives of compound 2-164 a) BnONH2 HCl or NH2OCH2CO2H (5 0 equiv.), Pyr/AcOH 5: 1 , 40 0C, 24 h, 20-35 %, b) PS-TsOH (10 0 equiv., 3 2 mmol.g 1), MeOH, 40 0C, 2.5 h, 77-80 %: c) Pφeπdme (1.1 equiv ), EDC (1.1 equiv ), HOBt (1.1 equiv ), DMF, 23 0C, 2 h, 75-80 %. [00288] In another embodiment, the pochonin oximes were prepared from cyclization of the pre-formed oximes as shown in Scheme 24. For example protected pre-formed oxime 1 was bound to Wang resin using yV.yV'-diisopiOpylcarbodiimide and dimethylaminopyridine. The allyl protecting group was removed and the phenol was functionalized under Mitsonubo conditions. This was followed by deprotection of the carboxylic acid and esterification with R3OH. Ring-closing was effected with the use of Grubbs II catalyst as described before to provide resin-bound oxime 5. Deprotection and removal from the resin was achived with trifluoroacetic acid to provide oxime 6. The carboxylic acid can be reacted with a variety of groups R4XH to provide the oximes 7, wherein X is oxygen, sulfur, amino or substituted amino. The oximes 7 were generally obtained as a 1 :1 mixture of E:Z isomers which could be separated by reverse-phase chromatograpy (C 18).
53412 v2 DC 107
Figure imgf000109_0001
Scheme 24
[00289] In another embodiment, the pochonin oximes were prepared by an alternate process which utilized a Mitsunobu cyclization to construct the macrocylic ring. Scheme 25 shows a non-limiting example for the formation of the macrocyle 2-al via a Mitsunobu esterification. Orcinol (compound 8) is oxidized with phosphoryl chloride in DMF to provide aldehyde 9, which is protected as the di-ethoxyrnethyl ether and subjected to oxidative chlorination with NaClO2 to produce carboxylic acid 10. The carboxylic acid is protected as a trimethylsilylethyl ester and treated with LDA and Weinreb amide 12 (Scheme 26) to provide the α,β-unsaturated ketone 13. Ketone 13 is reacted with carboxymethoxylamine hemihydrochloride in pyridine at 40° C to produce the corresponding oximes as a mixture of E and Z isomers. The oximes were converted to the desired amides 14 as a mixture of E and Z isomers by treatment with EDC and piperidine. Macrocyclization of precursor 14 to protected compound 15 was performed under Mitsunobu conditios by slow addition of DIAD to a solution of compound 14 and PhP3 in toluene. The phenol groups were deprotected by treatment with sulfonic acid resin at 40° C to produce 2a-l as a mixture of E and Z isomers. The mixture of E- and Z-isomers was separated to obtain the pure E and Z isomers 2a-l.
53412 v2 'DC 108
Figure imgf000110_0001
E 2a-1 EIZ 2a-1
Scheme 25: Cyclization under Mitsunobu Conditions
[00290] The diene Weireb amide 12 was prepared according to the process shown in Scheme 26. Trans-3-hexenedioic acid dimethyl ester 16 was reduced to the corresponding diolwith lithium aluminum hydride. The diol was mono-protected as the tert-butyldiphenylsilyl ether 17, and the free alcohol was converted to aldehyde 20 in three steps via the nitrile 19. Aldehyde 20 was then treated with Weireb amide ylide 21 to produce the diene Weireb amide 12, which was used to prepare compound 13 (Scheme 24).
53412 v2/ DC 109
Figure imgf000111_0001
66% 20
12
Scheme 26
[00291] Crystal structures of the E-izomer 2a- 1 and the related E-oxime 2a- 13, which lacks the chloro substituent on the aromatic ring, were obtained. The crystal structures are shown in Figures 1 and 2.
[00292] The solubilityof compounds 2a- 1 and 2a- 13 was determined. Both compounds were determined to be highly soluble in DMSO and DMA (> 5 g/mL). The good solubility of the compounds in DMSO and DMA enable formulations for intravenous or intraperitoneal administration. In one non-limiting example, a formulation of the oximes in DMSO/Tween 20/0.9% NaCl (10/5/85) was prepared. Biological Activity
Figure imgf000111_0002
(S)-2-150a A-5 (R)-2-150f A-6 (S)-2-150 A-7 (R)-2-147-3 A-8
Pochonin Analogs Selected for IC50 Determination
53412 DC 110 Examples
[00293] General Techniques. All reactions were carried out under a nitrogen atmosphere with dry (anhydrous) solvents under anhydrous conditions, unless otherwise noted. Anhydrous solvents were obtained by passing them through commercially available alumina column (Innovative Technology, Inc., Λ VA). All substituted polystyrene resins (100-200 mesh, 1 % DVB) were purchased from Novabiochem* or AldrichΛ\ The Grubbs" II catalyst was purchased from Materia Inc.κ Solid phase reactions were earned on a Quest Λ 210 or round bottom flasks and filtered in fritted funnels. Reactions were monitored by thin layer chromatography (TLC) carried out on 0.25 mm E. Merck* silica gel plates (60F-254) using UV light as visualizing agent and 10% ethanolic phosphomolybdic acid or vanillin solution and heat as developing agents. E. Merck'1 silica gel (60, particle size 0.040-0.063 mm) was used for flash column chromatography. PTLC (preparative thin layer chromatography) were carried out on 0.25 mm E. MerckΛ silica gel plates. NMR spectra were recorded on a Bruker Advance-400R instrument and calibrated by using residual undeuterated solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. LC-MS were recorded using an Agilent 1 100^ HPLC with a Bruker'1 micro-TOF instrument (ESI). Unless otherwise stated, a Supelco15 C8 (5 cm x 4.6mm, 5 μm particles) column was used with a linear elution gradient from 100% H2O (0.5% HCO2H) to 100% MeCN in 13 min at a flow rate of 0.5 ml/min. Unless otherwise stated, LDA was prepared at a concentration of 0.566 M by treating a solution of diisopropylamine (1.0 equiv.) in THF at -78 0C with ra-butyllithium (1.0 equiv.) and stirred for 30 min at this temperature before use. [00294] EXAMPLE 1 : General procedure for the synthesis of compounds 4. [00295] As depicted in Scheme 1, a solution of acid 2-95A or 2-95b (1.0 equiv), homoallylic alcohol (1.0 equiv) and tris-(3-chlorophenyl)phosphine (2.0 equiv) in anhydrous toluene (0.05 M) was treated at room temperature with PS-DEAD (2.5 equiv, 1.3 mmol g"1). After stirring for 10 min, the reaction mixture was filtered on silica and washed with hexane/EtOAc (10/1, 100 ml) and hexane/EtOAc (3/1, 100 ml). The 3/1 mixture was concentrated under reduced pressure to yield compound 3 (60-80%). Without further purification, compound 3 (1.0 equiv) and tetrabutylammonium iodide (catalytic amount) were dissolved in DMF (0.15 M) and treated with diisopropylethylamine (4.0 equiv) and (chloromethyl)ethyl ether (4.0 equiv). After stirring overnight at 80 0C, the reaction mixture was diluted with EtOAc and washed several times with a saturated NH4Cl solution. The organic phase was dried over MgSO4 and
53412 v2 DC ] ] ] concentrated under reduce pressure to yield compounds 4 (80-90%). Using this method, a variety of compounds 4 were prepared.
Figure imgf000113_0001
(/?)-2-217d
1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.04 (s, I H), 5.89 (ddt, J = 17.0, 10.5, 7.0 Hz, IH), 5.31 (s, 2H), 5.21 (s, 2H), 5.22-5.06 (m, 3H), 3.79 (q, J = 7.0 Hz, 2H), 3.72 (q, J = 7.0 Hz, 2H), 2.48-2.44 (m, 2H), 2.36 (s, 3H), 2.01 (qd, J = 12.4, 7.0 Hz, IH), 1.25 (t, J = 7.0 Hz, 3H), 1.23 (t, J = 7.0 Hz, 3H), 1.02 (d, J = 6.4 Hz, 3H), 1.01 (d, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 167.3, 154.0, 152.9, 134.8, 134.1 , 120.4, 1 17.5, 1 17.1, 101.5, 93.9, 93.4, 79.0, 64.6, 64.3, 35.6, 30.8, 18.4, 17.6, 17.5, 15.0 (x 2); HRMS (ESI-TOF) m/∑ 437.1574 ([M+N a""], C2i H3 ,06ClNa requires 437.1701).
The following non-limiting examples of compounds 4 were prepared.
53412 v2/DC 1 12
Figure imgf000114_0001
EXAMPLE 2: General procedure for the synthesis of compounds 2-118, 2-119 and 2-140.
[00296] As depicted in Scheme 1 , a solution of compound 2-110 or 2-117 (1.0 equiv) in anhydrous THF (0.2 M) was treated at -78 0C with freshly made LDA (2.0 equiv). Immediately after, the α,β-unsaturated Weinreb amide (S. V. Ley and I. R. Baxendale, Nat. Rev. Drug Discov., 1 :573 (2002)) was added to the cooled solution (1.0 equiv). The resulting
53412 v2, DC 1 13 mixture was then stirred for 10 min at -78 0C and quenched by addition of Amberlite* resin (20 equiv). Upon wanning up to room temperature, the reaction was filtered on a pad of silica and washed with EtOAc. Concentration under reduced pressure afforded the desired compound 5. This compound was used directly in the metathesis reaction without any further purification. When X = H, 20% of the corresponding 1 ,4-addition compound was observed and a fraction of the mixture was purified for characterization of compounds 5 and 7 (SiO2, 0- 20% EtOAc/cyclohexane gradient). Illustrative examples of compounds 5 and 7 follow; their characterization follows respectively in the text below each figure. Illustrative Compounds 5:
Figure imgf000115_0001
(i?)-2-119d
1H NMR (CDCl3, 400 MHz, 25 0C) δ 6.91 (dt, J = 15.8, 6.7 Hz, IH), 6.87 (d, J = 1.8 Hz, I H), 6.53 (d, J = 1.8 Hz, I H), 6.19 (d, J= 15.8 Hz, IH), 5.91-5.77 (m, 2H), 5.23 (s, 2H), 5.22 (s, 2H), 5.15-5.00 (m, 5H), 3.87 (s, 2H), 3.73 (q, J = 7.0 Hz, 4H), 2.45-2.40 (m, 2H), 2.35- 2.29 (m, 2H), 2.25-2.20 (m, 2H), 2.00- 1.92 (m, 1 H), 1.24 (t, J = 7.0 Hz, 6H), 1.00 (d, J = 2.3 Hz, 3H), 0.98 (d, J = 3.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 196.3, 167.6, 159.0, 156.2, 147.1 , 137.0, 135.0, 134.4, 129.5, 1 18.7, 1 17.2, 1 15.5, 1 1 1.0, 102.3, 93.3, 93.0, 78.8, 64.4, 64.3, 45.4, 35.8, 32.0, 31.7, 30.8, 18.5, 17.4, 15.0 (x 2); HRMS (ESI-TOF) m/z 51 1.2521 ([M+NV], C28H40O7Na requires 51 1.2666).
Illustrative Compound 7:
Figure imgf000115_0002
1H NMR (CDCl3, 400 MHz, 25 0C) δ 6.72 (s, IH), 6.57 (s, IH), 5.89-5.71 (m, 2H), 5.20-5.16 (m, 4H), 5.12-4.90 (m, 4H), 4.33 (t, J = 6.8 Hz, 2H), 3.69 (2 x q, J = 7.0 Hz, 4H), 3.57 (s,
53412 v2 'DC 1 14 3H), 3.13 (s, 3H), 2.69-2.64 (m, IH), 2.53-2.45 (m, 4H), 2.32 (m, 2H), 2.08-2.03 (m, 2H), 1.19 (t, J = 6.8 Hz, 6H), 1.01 (t, J = 6.5 Hz, 2H); HRMS (ESI-TOF) m/z 508.2873 ([M+H+], C27H42O8N requires 508.2905).
[00297] Using the procedure above, the following non-limiting examples of compounds 5 shown below were prepared.
Figure imgf000116_0001
Figure imgf000116_0002
Figure imgf000116_0003
[00298] The following non-limiting examples of compounds 2-140 were prepare according to the procedure described here.
53412 v2 DC 1 15
Figure imgf000117_0001
.0
O^ N \
2-14Og
EXAMPLE 3: General procedure for the metathesis reaction.
[00299] As depicted in Scheme 1 , a solution of crude 2-118 or 2-119 (or mixture 2-118/2- 119 and 2-140 when X = Cl), in anhydrous toluene (2 mM) was treated with Grubbs" second generation catalyst (0, 10 equiv) and heated at 80 0C for 12 h. The reaction was cooled down to room temperature and the mixture was filtered through a pad Of SiO2, washed with CH2CI2 followed by a mixture EtOAc/cyclohexane 1/1 and concentrated under reduced pressure. Purification by flash chromatography (SiO2, 0-25% EtOAc/cyclohexane gradient) afforded compounds 2-112 or 2-120 or 2-140 (60-85% over two steps). Illustrative examples of compounds 2-112 or 2-120 and 2-140 follow; their characterization follows respectively in the text below each figure.
53412 v2' DC 1 16 [00300] Illustrative Compounds 2-1 12/2-120:
Figure imgf000118_0001
1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.14 (s, IH), 6.72-6.66 (m, IH), 5.88 (d, J - 15.2 Hz, I H), 5.33-5.17 (m, 6H), 4.92-4.88 (m, IH), 4.21 (d, J = 17.0 Hz, IH), 3.92 (d, J = 17.0 Hz, I H), 3.79-3.67 (m, 4H), 2.33-2.17 (m, 5H), 2.07-1.96 (m, 2H), 1.23 (t, J = 7.0 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H), 1.00 (d, J = 5.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 195.7, 167.1 , 154.7, 154.4, 147.4, 133.7, 131.2, 128.8, 128.4, 1 19.7, 1 18.0, 102.7, 93.9, 93.5, 80.0, 64.8, 64.5, 44.1 , 32.3, 31.2, 30.7, 30.6, 18.3, 17.2, 15.0, 14.9; HRMS (ESI) m/z 517.1844 ([M+Na+], C26H35O7ClNa requires 517.1964); [α]25 D +21.3 (c 1.00, CHCl3).
Figure imgf000118_0002
1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.49-7.47 (m, 2H), 7.40-7.29 (m, 3H), 7.10 (s, IH), 6.84-6.77 (m, IH), 5.98 (d, J = 15.2 Hz, IH), 5.78 (d, J = 8.8 Hz, IH), 5.44-5.30 (m, 4H), 5.15 (d, J = 7.0 Hz, IH), 5.05 (d, J= 6.8 Hz, IH), 4.07 (d, J = 17.0 Hz, IH), 3.90 (d, J- 17.0 Hz, I H), 3.80 (d, J = 7.0 Hz, 2H), 3.60-3.51 (m, 2H), 2.68-2.62 (m, IH), 2.50-2.47 (m, IH), 2.38-2.29 (m, 2H), 2.14-2.02 (m, 2H), 1.25 (t, J = 7.0 Hz, 3H), 1.17 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 195.7, 166.7, 154.8, 154.2, 147.3, 140.7, 133.3, 132.1, 128.5, 128.3 (x 2), 128.2, 127.9, 127.7, 126.7 (x 2), 120.1 , 1 18.1 , 102.9, 93.9, 93.4, 77.4, 64.8, 64.4, 44.5, 40.5, 30.7, 15.0, 14.9; HRMS (ESI) m/z 551.1807 ([M+Naτ], C29H33O7ClNa requires 551.1680); [α]25 D -40.4 (c 0.79, CHCl3).
Figure imgf000118_0003
53412 v2'DC 1 17 1H NMR (CDCl3, 400 MHz, 250C) δ 7.39-7.33 (m, 4H), 7.31-7.27 (m, IH), 6.82 (s, IH), 6.82-6.75 (m, IH), 6.63 (s, IH), 6.02 (d, J= 16.4 Hz, IH), 5.35-5.29 (m, 2H), 5.27-5.20 (m, 5H), 4.16 (d, J= 14.6 Hz, IH), 3.79-3.70 (m, 4H), 3.52 (d, J - 14.6 Hz, IH), 3.37 (dd, J = 13.4,4.1 Hz, IH), 2.78 (dd, J= 13.5, 9.4Hz, IH), 2.37-2.12 (m, 5H), 2.06-2.02 (m, IH), 1.26 (t, J = 7.0 Hz, 3H), 1.24 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 250C) δ 197.6, 167.8, 159.2, 156.5, 149.0, 137.3, 135.5, 131.8, 129.9, 129.5 (x 2), 128.6 (x 2), 128.4, 126.7, 118.1, 109.9. 102.3, 93.5, 93.1, 75.8, 64.6, 64.4, 44.4, 41.0, 36.2, 31.0, 30.6, 15.0 (x 2); HRMS (ESI) m/z 531.2350 ([M+Na+], C30H36O7Na requires 531.2359); [α]25 D -24.1 (c 0.33, CHCl3).
Figure imgf000119_0001
1H NMR (CDCl3, 400 MHz, 250C) δ 7.56-7.54 (m, 2H), 7.41-7.29 (m, 3H), 6.89-6.82 (m, IH), 6.78 (d,J=2.3 Hz, IH), 6.61 (d, J= 1.8 Hz, IH), 6.06 (d, J= 16.4Hz, IH), 5.98 (dd, J = 11.7, 2.4Hz, IH), 5.53-5.51 (m, 2H), 5.20 (d, J = 7.0 Hz, IH), 5.17 (d, J = 6.4 Hz, IH), 5.07 (d, J= 7.0 Hz, IH), 4.96 (d, J= 7.0 Hz, IH), 4.20 (d, J= 14.6 Hz, IH), 3.73-3.68 (m, 2H), 3.54-3.45 (m, 3H), 2.71-2.66 (m, IH), 2.55-2.51 (m, IH), 2.38-2.32 (m, 2H), 2.23-2.06 (m, 2H), 1.22 (t, J = 7.0 Hz, 3H), 1.14 (t, J= 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 197.6, 167.4, 159.3, 156.6, 149.0, 140.8, 135.6, 132.2, 129.9, 128.5, 128.2 (x 2), 127.9, 126.9 (x 2), 117.9, 109.9, 102.3, 93.2, 93.0, 76.6, 64.4, 64.3, 44.4, 40.5, 31.0, 30.6, 15.0, 14.9; HRMS (ESI) m/z 517.2062 ([M+Na], C29H34O7Na requires 517.2197). [α]25 D -108.3 (c 1.00, CHCl3).
Illustrative Compounds 2-140:
Figure imgf000119_0002
O^N'α
53412 v2'DC 118 Mixture of 4 diastereoisomers: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 6.77 (s, IH), 6.52 (s, 0.5H), 6.46 (s, 0.5H), 5.59-5.37 (m, 2H), 5.21-5.18 (m, 4H), 5.09-4.92 (m, IH), 3.75-3.70 (m, 4H), 3.53-3.48 (m, 3H), 3.38-3.34 (m, IH), 3.19-3.10 (m, 3H), 2.65-2.47 (m, 3H), 2.29-2.04 (m, 6H), 1.89-1.72 (m, 2H), 1.31 -1.20 (m, 6H), 1.06-0.96 (m, 6H); HRMS (ESI-TOF) m/z 544.2907 ([M+Na~], C28H43O8NNa requires 544.2881).
Figure imgf000120_0001
Mixture of 4 diastereoisomers: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.51-7.42 (m, 2H), 7.38-7.31 (m, 3H), 6.73-6.70 (m, I H), 6.60-6.49 (m, IH), 6.45-6.31 (m, IH), 5.73-5.39 (m, 2H), 5.23-5.00 (m, 4H), 3.75-3.69 (m, 2H), 3.56-3.34 (m, 6H), 3.19-3.09 (m, 3H), 2.66-2.08 (m, 8H), 1.31-1.19 (m, 5H), 1.10-1.04 (m, 3H); HRMS (ESI-TOF) m/z 578.2715 ([M+Naτ], C3, H4108NNa requires 578.2724).
EXAMPLE 4: General procedure for the EOM deprotection to generate compounds deprotected-2-121 and 2-85.
[00301] As depicted in Scheme 17 for compounds 2-103/2-85, to a solution of the corresponding compound 2-120/2-112 or 2-140 (1 .0 equiv) in MeOH (0.03 M) was added PS-TsOH (10.0 equiv, 3.2 mmol/g) and the suspension was shaken at 40 0C for 1 to 4 h. The reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. Purification by flash chromatography (SiO2, 0-20% EtOAc/cyclohexane gradient) afforded the corresponding compound deprotected-2-140 or compound 2-103/2-85. (>90%). Illustrative examples of compounds deprotected-2-140 and of 2-103/2-85 follow; their characterization follows respectively in the text below each figure. [00302] Illustrative Compound Deprotected-2-140:
53412 v2'DC 19
Figure imgf000121_0001
Mixture of 4 diastereoisomers: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 1 1.54 (s, IH), 6.33 (d, J = 2.3 Hz, I H), 6.25 (s, I H), 5.53-5.51 (m, IH), 5.44-5.41 (m, IH), 5.1 1-5.08 (m, IH), 4.01 (d, J = 1 1.7 Hz, 2H), 3.45 (s, 3H), 3.11 (s, 3H), 2.83-2.73 (m, IH), 2.68-2.59 (m, IH), 2.27-2.20 (m, I H), 2.10-1.87 (m, 6H), 1.82-1.72 (m, IH), 1.01 -0.94 (m, 6H); HRMS (ESI-TOF) m/z 428.2109 ([M+Na~], C22H3 IU6NNa requires 428.2044).
Illustrative Compounds 2-103/2-85:
Figure imgf000121_0002
1H NMR (C6D6, 400 MHz, 25 0C) δ 12.31 (s, IH), 6.83 (s, IH), 6.74-6.67 (m, IH), 5.84 (bs, IH), 5.82 (d, J = 15.8 Hz, I H), 5.03-4.95 (m, IH), 4.88-4.86 (m, IH), 4.76-4.70 (m, IH), 4.40
(d, J = 17.6 Hz, I H), 4.15 (d, J = 17.5 Hz, IH), 2.40-2.34 (m, IH), 2.22-2.18 (m, IH), 1.87-
1.65 (m, 4H), 1.53- 1.48 (m, 1 H), 0.92 (d, J = 6.4 Hz, 3H), 0.66 (d, J = 7.0 Hz, 3H); 13C NMR
(C6D6, 100 MHz, 25 0C) δ 193.7, 164.2, 156.8, 145.8, 137.2, 131.8, 129.3, 126.3, 1 15.3,
107.9, 103.6, 82.1 , 46.4, 33.3, 30.9, 30.7, 28.8, 20.1 , 18.5, 18.3; HRMS (ESI-TOF) m/z 401.1 170 ([M+Nal, C20H23ClO5Na requires 401.1 126); [α]25 D -35.6 (c 0.52, CHCl3).
Figure imgf000121_0003
1H NMR (C6D6, 400 MHz, 25 0C) δ 12.0 (bs, IH), 7.32-7.29 (m, 3H), 7.19-7.15 (m, 2H), 6.86-6.79 (m, IH), 6.51 (d, J = 2.4 Hz, IH), 6.27-6.25 (m, IH), 6.1 1 (d, J= 2.4 Hz, IH), 6.02 (d, J= 15.8 Hz, IH), 5.49 (s, IH), 5.17-5.10 (m, IH), 4.97-4.90 (m, IH), 4.40 (d, J= 16.4 Hz,
53412 v2/ DC 120 IH).3.97 (d, J= 17.2 Hz, IH), 2.83-2.76 (m, IH), 2.45-2.38 (m, IH), 1.89-1.78 (m, 2H), 1.67-1.58 (m, 2H); 13C NMR (C6D6, 100 MHz, 250C) δ 196.5, 169.6, 166.1, 161.3, 146.0, 140.5, 138.8, 132.1, 130.0, 128.6 (x 2), 127.3, 126.6 (x 2), 126.3, 112.2, 105.9, 103.0, 77.1, 48.6, 38.4, 30.9, 30.3; HRMS (ESI) m/z 401.1271 ([M+Na"], C23H22O5Na requires 401.1359); [α]25 D -10.3 (c 0.25, CHCl3).
Figure imgf000122_0001
1H NMR (CDCl3, 400 MHz, 250C) δ 12.43 (s, IH), 6.74 (d, J= 1.7 Hz, IH), 6.73-6.65 (m, IH), 6.48 (d, J= 1.7 Hz, IH), 5.92 (d, J= 15.8 Hz, IH), 5.12-5.00 (m, 2H), 4.91-4.80 (m, IH), 4.19 (d, J= 17.0 Hz, IH), 3.84 (d, J= 16.4 Hz, IH), 2.77 (m, IH), 2.64-2.57 (m, IH), 2.01-1.97 (m, IH), 1.89-1.70 (m, 3H), 1.61-1.56 (m, 2H), 1.30-1.21 (m, 2H), 0.90 (t, J= 6.7 Hz, 3H); 3C NMR (CDCl3, 100 MHz, 250C) δ 197.5, 169.9, 165.6, 160.6, 147.5, 140.2, 131.9, 129.5, 127.0, 112.8, 106.1, 102.9,76.2,48.7,35.7,34.3,31.1,29.7, 19.4, 13.8; HRMS (ESI-TOF) m/z 367.1330 ([M+Naτ], C20H24O5Na requires 367.1521); [α]25 D +21.6 (c 0.36, CHCl3).
Figure imgf000122_0002
!H NMR (CD3OD, 400 MHz, 250C) δ 6.78-6.71 (m, IH), 6.29 (d, J= 2.4 Hz, IH), 6.22 (d, J = 2.0 Hz, IH), 5.87 (d, J= 15.5 Hz, IH), 5.37-5.23 (m, 3H), 4.01 (d, J= 17.2 Hz, IH), 3.92 (d, J= 17.0 Hz, IH), 2.67-2.61 (m, IH), 2.29-2.15 (m, 5H), 1.31 (d, J= 6.4 Hz, 3H); 13C NMR (CD3OD, 100 MHz, 250C) δ 198.5, 169.8, 164.2, 162.3, 148.4, 139.1, 131.6, 129.6, 127.3, 111.7, 101.7, 72.0, 47.7, 36.8, 30.8, 30.7, 17.4, (1 quartenary carbon is not visible); HRMS (ESI) m/z 339.1141 ([M+Na], C18H20O5Na requires 339.1203). [α]25 D -45.1 (c 0.27, CHCl3).
53412 \2 DC 121
Figure imgf000123_0001
!H NMR (CD3OD, 400 MHz, 25 0C) δ 6.74-6.68 (m, I H), 6.48 (s, I H), 5.86 (d, J = 15.2 Hz, IH), 5.31 -5.25 (m, 2H). 4.39 (t, J = 5.3 Hz, 2H), 4.27 (s, 2H), 2.43-2.40 (m, 2H), 2.25 (m, 4H); 13C NMR (CD3OD, 100 MHz, 25 0C) δ 196.9, 170.1 , 161.9, 158.1 , 147.8, 135.9, 130.9, 130.2, 129.9, 1 15.2, 107.3, 102.4, 65.9, 46.2, 31.3, 30.9, 30.5; HRMS (ESI) m/z 337.0797 ([M+Hx], C7H18O5Cl requires 337.0837).
EXAMPLE 5: General procedure for the synthesis of compounds 2-141. [00303] As depicted in Scheme 17, to a solution of corresponding compound 2-120/2-112 (1.0 equiv) in MeOH (0.03 M) at 0 0C was added BER-resin (Borohydride on Amberlite*, 1.0 equiv, 2.5 mmol g" 1) and the reaction was stirred over 12 h. The reaction was then filtered and concentrated under reduced pressure. Purification by flash chromatography (SiO?, 0-20% EtOAc/cyclohexane gradient) afforded 2-141 (-60%) as a mixture of two diastereoisomers (1 : 1 ). An illustrative example of compound 2-141 follows; its characterization data are in the text below the figure.
Illustrative Compound 2-141:
Figure imgf000123_0002
1H NMR (CD3Cl, 400 MHz) δ 7.05 (s, IH), 6.99 (s, IH), 5.64-5.57 (m, 2H), 5.54-5.53 (m, 2H), 5.49-5.35 (m, 6H), 5.31 -5.28 (m, 4H), 5.24-5.16 (m, 4H), 5.13-5.08 (m, IH, 35'), 4.68 (m, I H, 35"), 4.56 (m, I H, 35), 3.81-3.69 (m, 8H), 3.25 (dd, J = 13.9, 8.0 Hz, IH, 35), 3.19 (dd, J = 13.7, 4.8 Hz, IH, 35"), 3.1 1 (dd, J = 13.5, 10.1 Hz, IH, 35'), 2.90 (dd, J= 13.9, 5.12 Hz, I H, 35), 2.35 (m, 9H), 2.09-1.95 (m, IH), 1.80-1.70 (m, 2H), 1.39 (d, J = 2.9 Hz, 3H, 35), 1.37 (d, J = 3.2 Hz, 3H, 35"), 1.24 (2 x q, J = 6.9 and 5.0 Hz, 12H, 35 + 35'); HRMS (ESI) m/z 491.1729 ([M+Na~], C24H33ClO7Na requires 491.1807).
53412 v2, DC 122 EXAMPLE 6: General procedure for the synthesis of compounds 2-142, [00304] As depicted in Scheme 17, to a solution of the corresponding compound 2-141 (1.0 equiv) in MeOH (0.02 M) was added PS-TsOH (10.0 equiv, 3.2 mmol g"1) and the suspension was shaken at 40 0C for 4 h. The reaction mixture was then filtered and the methanolic solution concentrated under reduced pressure. Purification by preparative TLC (SiO2, 25% EtOAc/cyclohexane) afforded 2-142 (-90%) as a mixture of two diastereoisomers (1 :1). An illustrative example of compound 2-142follows; its characterization data are in the text below the figure.
Illustrative Compound 2-142:
Figure imgf000124_0001
1H NMR ((CD3)2CO, 400 MHz) δ 12.30 (s, 2H), 1 1.43 (s, 2H), 6.75 (s, 2H), 6.00 (bdd, J =
6.4, 6.2 Hz, IH), 5.97 (bdd, J = 6.4, 6.2 Hz, I H), 5.97 (bd, J = 6.7 Hz, I H), 5.77 (bd, J = 6.7 Hz, I H), 5.57-5.48 (m, 4H), 5.18-5.14 (m, 2H), 3.38-3.28 (m, 3H), 3.02 (dd, J = 16.1 , 10.5 Hz, I H), 2.41 -2.09 (m, 12H), 1.1 1 (d, J= 6.2 Hz, 6H); HRMS (ESI) m/z 375.1029 ([M+Na+], C18H21ClO5Na requires 375.0970).
EXAMPLE 7: General procedure for the synthesis of compounds 2-143. [00305] As depicted in Scheme 17, to a solution of the corresponding compound 2-141 (1.0 equiv) in DMF (0.02 M) were added Ac2O (1.2 equiv), morpholinomethyl polystyrene (1.2 equiv, 3.2 mmol g"1) and DMAP (0.05 equiv) at 23 0C and the mixture was stirred for 30 min, followed by TLC until consumption of the starting material. Then, the resin was filtered and the organic phase was concentrated under reduced pressure. Purification by PTLC (SiO2, 20% EtOAc/cyclohexane) afforded corresponding 2-143 (-80%) as a mixture of two diastereoisomers 1 : 1 : An illustrative example of compound 2-143follows; its characterization data are in the text below the figure.
53412 vZ DC 123 Illustrative Compound 2-143:
Figure imgf000125_0001
1H NMR (CD3Cl, 400 MHz) δ 7.04 (s, I H), 7.01 (s, IH), 5.86 (dd, J= 15.0, 6.9 Hz, IH), 5.67 (dd, J = 12.4, 6.2 Hz, IH), 5.60-5.54 (m, 4H), 5.48 (dd, J - 7.2, 7.2 Hz, I H), 5.41 -5.34 (m, 3H), 5.32-5.30 (m, 4H), 5.28-5.23 (m, 2H), 5.21 (dd, J = 1 1.0, 6.7 Hz, 2H), 5.17 (dd, J = 1 1.8, 6.9 Hz, 2H), 3.81 -3.69 (m, 8H), 3.43 (dd, J = 14.2, 7.5 Hz, I H), 3.23-3.15 (m, 2H), 2.85 (dd, J = 13.9, 5.4 Hz, IH), 2.30-2.17 (m, 8H), 2.12 (s, 3H), 2.06 (s, 3H), 1.95-2.00 (m, 4H), 1.39 (2 x d, J = 5.6 Hz, 6H), 1.24 (m, 12H); HRMS (ESI) m/z 533.1864 ([M+Naτ], C26H35ClO8Na requires 533.1913).
EXAMPLE 8: General procedure for the synthesis of compounds 2-144. [00306] As depicted in Scheme 17, to a solution of corresponding compound 2-143 (1.0 equiv) in MeOH (0,02 M) was added PS-TsOH (10.0 equiv, 3.2 mmol/g) and the suspension was shaken at 40 0C for 4 h. The reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. Purification by PTLC (SiO2, 20% EtOAc/cyclohexane) afforded compounds 2-144 (-60% yield). An illustrative example of compound 2-144 follows; its characterization data are in the text below the figure. Illustrative Compound 2-144:
Figure imgf000125_0002
Mixture of diastereoisomers 2:1 : 1H NMR (CD3Cl, 400 MHz) δ 12.6 (s, IH), 12.12 (s, 0.5H), 6.93 (d, J = 8.7 Hz, 0.5H), 6.66 (s, IH), 6.64 (s, 0.5H), 6.62-6.60 (m, IH), 6.10-6.05 (m, 3H), 5.47-5.33 (m, 4.5H), 2.60-2.53 (m, 1.5H), 2.26-2.02 (m, 7.5H), 1.44 (d, J = 6.2 Hz, 1.5H), 1.43 (d, J - 6.4 Hz, 3H); HRMS (ESI) m/z 357.0898 ([M+Na+], Ci8Hi9ClO4Na requires 357.0864).
53412 v2/DC 124 EXAMPLE 9: General procedure for the synthesis of compounds 2-145. [00307] As depicted in Scheme 18, to a solution of corresponding compound 2-85 (1.0 equiv) in methanol (0.03 M) was added sulfamic acid resin (10.0 equiv) and the suspension was stirred for 15h at 4O0C with. The reaction was then filtered, the resin washed several times with CH2Cl?. Concentration under reduced pressure followed by purification on PTLC (Hexane/EtOAc : 1/1 ) afforded desired compounds 2-145as a mixture diastereoisomers (2: 1). An illustrative example of compound 2-145follows; its characterization data are in the text below the figure.
Illustrative Compound 2-145:
Figure imgf000126_0001
1H NMR (C6D6, 400 MHz, 25 0C) δ 12.28 (s, 0.4H), 1 1.91 (s, 0.6H), 7.21 -7.1 1 (m, 5H), 6.62 (s, I H), 6.03-6.01 (m, I H), 5.58 (bs, I H), 5.38-5.33 (m, IH), 5.27-5.20 (m, I H), 4.76 (d, J = 17.5 Hz, 0.6H), 4.02 (d, J = 17.0 Hz, 0.4H), 4.18 (d, J = 18.1 Hz, 0.6H), 4.09 (d, J = 17.0 Hz, 0.4H), 3.87 (bs, 0.4H), 3.81 (bs, 0.6H), 3.15 (s, 1.8H), 3.12 (s, 1.2H), 2.83-2.78 (m, IH), 2.45-2.30 (m, 2H), 2.18-2.16 (m, I H), 2.02-1.97 (m, 2H), 1.79-1.72 (m, 2H) ; HRMS (ESI- TOF) m/z 467.1366 ([M+Na+], C24H25O6ClNa requires 467.1232).
EXAMPLE 10: General procedure for the synthesis of compounds 2-146. [00308] As depicted in Scheme 19, to a solution of corresponding compound 2-103/2-85 (1.0 equiv) in CH2Cl2/Ac0H 10/1 (0.08 M) (polystyrylmethyl)trimethylamrnonium cyanoborohydride (2.0 equiv, 3.5 mmol g*1) was added at 23 0C and the reaction was monitored by TLC until the starting material had been consumed (4 h). Then, the resin was filtered and the organic phase was concentrated under reduced pressure. Purification by PTLC (SiO2, 30% EtOAc/cyclohexane) afforded compounds 2-146 (50-60%). An illustrative example of compound 2-146follows; its characterization data are in the text below the figure.
5.3412 v2/ DC 125 Illustrative Compound 15:
Figure imgf000127_0001
1H NMR (CD3Cl, 400 MHz) δ 1 1.75 (s, I H), 6.65 (s, IH), 5.48 (m, 2H), 5.49 (ddt, J = 6.1 , 3.5, 2.9 Hz, IH), 4.53 (d, J = 17.5 Hz, I H), 4.04 (d, J = 17.7 Hz, I H), 2.61-2.54 (m, 2H), 2.48-2.28 (m, 3H), 2.19-2.14 (m, IH), 2.08-1.99 (m, IH), 1.72-1.61 (m, 3H), 1.41 (d, J = 6.4 Hz, 3H); HRMS (ESI) m/z 375.1050 ([M+Na+], Ci8H2 I ClO5Na requires 375.0970).
EXAMPLE 1 1 : General procedure for the synthesis of compounds 2-147. [00309] As depicted in Scheme 19, to a solution of corresponding compound 2-103/2-85 (1.0 equiv), in THF (0.05 M) were added in a sequential manner the corresponding alcohol (2.0 equiv), triphenylphosphine (2.0 equiv) and ethoxycarbonylazocarboxymethyl polystyrene (2.0 equiv, 1.3 mmol g"1). The reaction mixture was shaken at room temperature for 8 hours, and then, the resin was filtered and the filtrates were directly purified by PTLC (SiOo, 10% EtOAc/cyclohexane) to afford a mixture of compound 2-147 along with the bis-allylated product(78 %). An illustrative example of compound 2-147 follows; its characterization data are in the text below the figure.
Illustrative Compound 2-147:
Figure imgf000127_0002
Mixture with the corresponding bis-allylated compound (1 : 1): H NMR (CD3Cl, 400 MHz) δ 1 1.83 (s, IH), 6.82 (ddd, J = 15.7, 8.2, 4.6 Hz, I H), 6.72-6.65 (m, IH), 6.46 (s, IH), 6.41 (s, I H), 6.09-5.98 (m, 3H), 5.82 (d, J = 15.7 Hz, IH), 5.46-5.16 (m, 8H), 4.57-4.54 (m, 3H), 4.51-4.49 (m, 3H), 4.19 (d, J = 17.5 Hz, IH), 4.1 1 (d, J= 14.6 Hz, IH), 3.78 (d, J= 17.0 Hz, IH), 3.51 (d, J = 14.2 Hz, IH), 2.76-2.69 (m, IH), 2.38-2.05 (m, HH), 1.42 (d, J = 6.2 Hz, 3H), 1.35 (d, J = 6.3 Hz, 3H); mono-allylated compound HRMS (ESI) m/z 413.1 103 ([M+Na~], C21H23ClO5Na requires 413.1 132); bis-allylated compound HRMS (ESI) m/z 453.1422 ([M+Na+], C24H27ClO5Na requires 453.1449).
53412 v2/ DC 126 EXAMPLE 12: General procedure for the synthesis of compounds 2-148. [00310] As depicted in Scheme 19, to a solution of the corresponding compound 2-103/2-85 (1.0 equiv) in CH2Cl2 (0.05 M) was added TBD-methyl polystyrene (2.0 equiv, 2.9 mmol g"1) and the corresponding alkyl bromide or chloride (BrCH2COO1Bu, EOMCl) (0.9 equiv) at 23 0C and the mixture was shaken for 3 h. The resin was then filtered and the filtrates were concentrated under reduced pressure. Purification by PTLC (SiO2, 30% EtOAc/cyclohexane) afforded corresponding compound 2-148 (> 90%). Illustrative examples of compound 2-148 follows; their characterization data are presented respectively in the text below each figure. Illustrative Compounds 2-148:
Figure imgf000128_0001
1H NMR (CD3Cl, 400 MHz) δ 1 1.84 (s, IH), 6.69 (m, IH), 6.41 (s, IH), 5.76 (d, J= 15.0 Hz, I H), 5.43 (m, I H), 5,26 (ddd, J = 15.0, 9.1 , 4.8 Hz, IH), 5.18-5.1 1 (m, IH), 4.65 (s, 2H), 4.33 (d, J = 17.7 Hz, IH), 4.16 (d, J = 17.5 Hz, IH), 2.65-2.58 (m, IH), 2.37-2.34 (m, 2H), 2.25- 2.21 (m, IH), 2.12-2.01 (m, 2H), 1.53 (s, 9H), 1.34 (d, J = 6.5 Hz, 3H); HRMS (ESI) m/z 487.1498 ([M+Naτ], C24H29ClO7Na requires 487.1494).
Figure imgf000128_0002
1H NMR (C6D6, 400 MHz, 25 0C) δ 1 1.76 (s, I H), 6.86 (s, I H), 6.70 (dt, J = 14.9, 7.3 Hz, I H), 5.77 (d, J = 15.8 Hz, IH), 5.46-5.42 (m, I H), 5.37 (s, 2H), 5.30-5.19 (m, 2H), 4.34 (d, J = 17.6 Hz, I H), 4.16 (d, J = 18.1 Hz, I H), 3.80 (q, J = 7.0 Hz, 2H), 2.66-2.59 (m, IH), 2.37- 2.34 (m, 2H), 2.26-2.21 (m, IH), 2.13-2.06 (m, 2H), 1.34 (d, J = 6.4 Hz, 3H), 1.27 (t, J = 7.0 Hz, 3H); HRMS (ESI) m/z ([M+Na], C21H25O6ClNa requires 431.1237).
EXAMPLE 13: General procedure for the synthesis of compounds 2-149. [00311] As depicted in Scheme 19, to a solution of compound 2-103/2-85 (1.0 equiv) in acetone/H20 10/1 (0.05 M) was added OsO4 (0.1 equiv) followed by NMO (1.0 equiv) at 23 0C and the mixture was stirred for 1 h. The crude mixture was filtered through a plug of silica,
53412 v2'DC 127 concentrated and purified by PTLC (SiO2, 30% EtOAc/cyclohexane) to afford 2-149 (>70%) as a mixture of two diastereoisomers. An illustrative example of compound 2-149 follows; its characterization data are in the text below the figure. Illustrative Compound 2-149:
Figure imgf000129_0001
1H NMR (CD3OD, 400 MHz) δ 7.19 (m, IH), 6.89-6.81 (m, IH), 6.52 (s, IH), 6.47 (s, I H), 6.20 (d, ./ = 16.1 Hz, IH), 6.04 (d, J = 15.6 Hz, IH), 5.54-5.49 (m, IH), 5.43-5.36 (m, I H), 4.50 (d, ./ = 17.7 Hz, I H), 4.46 (d, J = 17.7 Hz, IH), 4.39 (d, J = 17.2 Hz, I H), 4.07 (d, J = 17.2 Hz, I H), 3.80-3.64 (m, 2H), 3.51 -3.46 (m, 2H), 2.62-2.58 (m, 2H), 2.39-2.30 (m, 2H), 2.27-2.18 (m, 2H), 2.08-2.98 (m, 2H), 2.00-1.85 (m, 4H), 1.44 (d, J = 6.4 Hz, 6H); HRMS (ESI) m/z 407.1031 ([M+Na*], C|8H2iC107Na requires 407.0868).
EXAMPLE 14: General procedure for the synthesis of compounds 2-150. [00312] As depicted in Scheme 19, to a solution of compound 2-103/2-85 (1.0 equiv) in CH3CN (0.03 M) at 0 0C was added freshly made DMDO (1.2 equiv, 0.04 M in acetone) and the mixture was stirred for 30 min. After evaporation of the solvents under reduced pressure, purification by PTLC (SiO2, 30% EtOAc/cyclohexane) afforded epoxides 2-150 (>90%) as a mixture of two diastereoisomers. Illustrative examples of compound 2-150 follow; their characterization data are presented respectively in the text below each figure. Illustrative Compounds 2-150:
Figure imgf000129_0002
1H NMR (CDCl3, 400 MHz) δ 1 1.84 (s, 2H), 6.94-6.82 (m, 2H), 6.69 (s, IH), 6.66 (s, IH), 6.23 (d, J= 17.1 Hz, I H), 6.1 1 (dd, J = 13.2, 1.6 Hz, I H), 5.39 (tdd, J= 7.5, 3.2, 2.7 Hz, IH), 5.32 (m, I H), 4.53 (d, J = 17.7 Hz, 2H), 4.27 (d, J = 17.7 Hz, 2H), 2.79-2.76 (m, IH), 2.74- 2.69 (m, IH), 2.58 (m, IH), 2.56 (m, IH), 2.47-2.24 (m, 8H), 2.13-2.08 (m, IH), 2.05-2.03 (m, IH), 1.91 (dd, J = 4.3, 4.3 Hz, IH), 1.87 (dd, J = 4.3, 4.3 Hz, IH), 1.51 (d, J = 6.4 Hz,
53412 \2/DC 128 3H), 1.35 (d, J= 6.4 Hz, 3H); HRMS (ESI) m/z 389.0724 ([M+Na+], C18H19ClO6Na requires 389.0762).
Figure imgf000130_0001
1H NMR (C6D6, 400 MHz, 250C) δ 11.56 (2 x s, 2H), 6.92-6.82 (m, 2H), 6.71 (s, IH), 6.67 (s, IH), 6.20 (m, 3H), 6.06 (d, J= 15.8 Hz, IH), 5.11 (bs, IH), 5.94 (m, IH), 4.46 (2 x d, J = 18.1 Hz, 2H), 4.20 (2 x d, J= 18.1 Hz, 2H), 2.72-2.70 (m, 2H), 2.53-2.48 (m, 4H), 2.38-2.35 (m, 3H), 2.25-2.13 (m, 5H), 1.84-1.77 (m, 2H), 1.05-1.01 (m, 6H), 0.91-0.88 (m, 3H), 0.86- 0.84 (m, 3H); HRMS (ESI) m/z 417.1128 ([M+Na~], C20H23O6ClNa requires 417.1075).
Figure imgf000130_0002
1H NMR (C6D6, 400 MHz, 250C) δ 11.80 (2 x s, 2H), 7.43-7.18 (m, 10H), 7.03-6.95 (m, 2H), 6.69 (s, IH), 6.61 (s, IH), 6.30 (d, J= 16.4Hz, IH), 6.21 (d,J= 15.8Hz, IH), 6.15-6.10 (m, IH), 6.03 (d, J= 11.1 Hz, IH), 4.84(2 x d, J= 18.1 Hz, 2H), 4.41 (2 x d, J= 17.6 Hz, 2H), 2.68-2.60 (m, 4H), 2.41-2.27 (m, 8H), 1.83-1.76 (m, 4H); HRMS (ESI) m/z 451.1028 ([M+Na], C23H2IO6ClNa requires 451.0919).
Figure imgf000130_0003
Major isomer : 1H NMR (C6D6, 400 MHz, 250C) δ 11.94 (s, IH), 7.36-7.28 (m, 5H), 6.95- 6.88 (m, IH), 6.42 (s, IH), 6.22 (s, IH), 6.11 (d, J = 15.8 Hz, IH), 5.47 (m, IH), 5.41 (bs, IH), 4.43 (d, J= 17.5 Hz, IH), 3.56 (d, J = 17.6 Hz, IH), 3.19 (dd, J= 13.7, 6.0 Hz, IH),
53412 v2/DC 129 3.03 (dd, J= 13.7, 7.9 Hz, IH), 2.87 (bs, IH), 2.70-2.28 (m, 4H), 2.03-1.93 (m, 2H); HRMS (ESI) m/z 431.1578 ([M+Na], C24H24O6Na requires 431.1465).
Figure imgf000131_0001
1H NMR (C6D6, 400 MHz, 250C) δ 11.98 (s, IH), 6.91-6.83 (m, IH), 6.43 (d, J = 2.3 Hz, IH), 6.24 (d, J =2.4 Hz, IH), 6.11 (d,J= 15.8Hz, IH), 5.35 (bs, IH), 5.29 (m, IH), 4.52 (d, J= 17.5 Hz, IH), 3.63 (d,J= 17.5 Hz, IH), 2.77 (m, 2H), 2.57-2.52 (m, 2H), 2.46-2.27 (m, 2H), 2.14-2.10 (m, IH), 1.93-1.88 (m, IH), 1.48 (d, J= 6.4 Hz, 3H); other isomer: 1H NMR (C6D6, 400 MHz, 250C) δ 11.67 (s, IH), 6.89-6.83 (m, IH), 6.40 (d, J= 2.4 Hz, IH), 6.24 (d, J= 2.9 Hz, IH), 6.21 (d, J= 16.4Hz, IH), 5.37 (bs, IH), 5.22 (m, IH), 4.20 (d, J= 17.0Hz, IH), 4.06 (d, J= 17.0Hz, IH), 2.74 (m, 2H), 2.57-2.20 (m, 4H), 1.80-1.76 (m, IH), 1.68-1.60 (m, IH), 1.37 (d, J= 6.4 Hz, 3H); HRMS (ESI) m/z 355.1249 ([M+Na], C18H20O6Na requires 355.1152).
EXAMPLE 15: General procedure for the synthesis of compounds 2-151. [00313] As depicted in Scheme 20, to a solution of compound 2-120 (1.0 equiv) in dioxane (0.05 M) at 230C was added HClCOnc. (20 equiv), and the mixture was stirred for 3 h. After that time the reaction was filtered through a plug of silica gel, the solvents were evaporated under reduced pressure, and purified by PTLC (SiO2, 30% EtOAc/cyclohexane) to afford compound 2-151 (>75%) as a mixture of two diastereoisomers. Illustrative examples of compound 2-151 follow; their characterization data are presented respectively in the text below each figure.
Illustrative Compounds 2-151:
Figure imgf000131_0002
1H NMR (CDCl3, 400 MHz) δ 12.11 (s, IH), 11.78 (s, IH), 6.51 (s, IH), 6.43 (s, IH), 6.41
(d, J= 2.4 Hz, IH), 6.37 (d, J= 2.7 Hz, IH), 6.21 (d, J = 2.4 Hz, IH), 6.11 (d, J= 2.4 Hz, IH), 5.59-5.51 (m, 3H), 5.40-5.32 (m, 3H), 4.54 (d, J= 17.2 Hz, IH), 4.42 (d,J= 17.2 Hz,
53412 v2 DC 130 IH), 3.60 (d, J = 17.2 Hz, IH), 3.45 (d, J = 17.0 Hz, I H), 3.28 (dd, J = 18.5, 9.4 Hz, IH), 3.1 1 (dd, J = 13.7, 6.2 Hz, I H), 3.07 (dd, J = 13.4, 4.6 Hz, IH), 2.76 (dd, J = 19.0, 6.2 Hz, IH), 2.62 (ddd, J= 15.5, 8.8, 4.0 Hz, IH), 2.54 (ddd, J= 15.3, 6.2, 3.2 Hz, IH), 2.40-2.26 (m, 4H), 2.25-2.13 (m, 4H), 2.03-1.91 (m, 2H), 1.42 (d, J = 6.4 Hz, 3H), 1.40 (d, J= 6.4 Hz, 3H); HRMS (ESI) m/z 375.0928 ([M+Na+], Ci8H2IClO5Na requires 375.0970).
Figure imgf000132_0001
1H NMR (C6D6, 400 MHz, 25 0C) δ 1 1.76 (s, 0.5H), 1 1.36 (s, 0.5H), 7.40-7.29 (m, 5H), 6.65 (s, 0.5H), 6.62 (s, 0.5H), 6.18 (t, J = 5.8 Hz, IH), 6.14 (s, 0.5H), 6.12 (s, 0.5H), 5.67-5.62 (m, IH), 5.55-5.49 (m, I H), 4.93 (d, J = 18.1 Hz, 0.5H), 4.80 (d, J = 17.1 Hz, 0.5H), 4.58-4.56 (m, I H), 4.38 (d, J= 18.1 Hz, 0.5H), 4.18 (d, J= 17.1 Hz, 0.5H), 3.33-3.27 (m, IH), 3.10 (dd, J= 18.4, 3.8 Hz, 0.5H), 2.84-2.68 (m, 2.5H), 2.42-2.32 (m, 2H), 2.23-2.17 (m, I H), 2.13-2.04 (m, I H); HRMS (ESI-TOF) m/z 471.0754 ([M+Na+], C23H22O5Cl2Na requires 471.0737).
EXAMPLE 16: General procedure for the elimination of β-Cl from compounds 2-151. [00314] As depicted in Scheme 20, to a solution of compound 2-151 (95 mg, 270 μmol) in CH2Cl2 (5 ml) at 23 0C PS-TBD (51 mg, 2.6 mmol/g) was added, and the mixture was stirred for 8 hours. After that time the reaction was filtered, the solvents were evaporated under reduced pressure, and purification by flash chromatography (SiO2, 0-30% EtOAc/cyclohexane gradient) afforded 2-103 (X = Cl, R = Me) (84 mg, 98%).
EXAMPLE 17: General procedure for the synthesis of compounds 2-153.
[00315] As depicted in Scheme 20, to a solution of compound 2-103 (X = Cl, R = Me) (12.9 mg, 40.8 μmol) in CH2Cl2 (1 ml) at 23 0C DHP (3.7 μL, 40.8 μmol) and PS-TsOH (12.7 mg, 40.8 μmol, 3.2 mmol/g) were added, and the mixture was stirred for 5 hours. After that time the reaction was filtered and the solvents were evaporated under reduced pressure. Purification by PTLC (SiO2, 30% EtOAc/cyclohexane) afforded 2-153 (13.8 mg, 85%) as a mixture of two diastereoisomers. An illustrative example of compounds 2-153 follows; its characterization data are in the text below the figure.
534 I2 V2/ DC 131 Illustrative Compound 2-153:
Figure imgf000133_0001
1H NMR (CDCl3, 400 MHz) δ 12.33 (s, I H), 12.1 1 (s, I H), 9.45 (s, I H), 9.40 (s, I H), 6.67, (m, 2H), 6.28 (2 x s, 2H), 5.83 (d, J = 13.2 Hz, IH), 5.79 (d, J= 12.9 Hz, IH), 5.35-5.30 (m, 3H), 5.27-5.22 (m, 3H), 5.06 (bd, J = 8.2 Hz, 2H), 4.10 (d, J = 17.5 Hz, 2H), 3.90-3.85 (m, I H), 3.80-3.76 (m, IH), 3.65 (d, J = 17.7 Hz, 2H), 3.57-3.52 (m, 2H), 3.46-3.41 (m, 2H), 2.77-2.71 (m, 3H), 2.53-2.49 (m, 3H), 2.36-2.29 (m, 4H), 2.24-1.56 (m, 12H), 1.31 (d, J = 6.4 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H); HRMS (ESI) m/z 423.1778 ([M+Naτ], C23H28O6Na requires 423.1778.
EXAMPLE 18: General procedure for the synthesis of compounds 2-154, [00316] As depicted in Scheme 20, to a solution of corresponding compound 2-120 (1.0 equiv) in pyridine/AcOH (5/1 , 0.03 M) was added the corresponding hydroxylamine (5.0 equiv) and the mixture was heated up to 40 0C. After stirring overnight the solvents were evaporated under reduced pressure with SiO2. Elution of the compound over a short path of SiO2 with a mixture of 30% EtOAc/cyclohexane afforded after evaporation 2-154 (-99%) as a mixture of two diastereoisomers cis/trans). An illustrative example of compound 2-154 follows; its characterization data are in the text below the figure. Illustrative Compound 2-154:
Figure imgf000133_0002
1H NMR (CDCl3, 400 MHz) δ 7.50-7.25 (m, 10H), 6.82 (s, IH), 6.75 (s, IH), 6.66 (s, IH), 6.48 (s, I H), 6.24-6.1 1 (m, 2H), 6.1 1-6.05 (m, 2H), 5.45-5.38 (m, 4H), 5.34-5.31 (m, 14 H), 4.50 (d, J = 17.2 Hz, IH), 3.38-3.65 (m, 8H), 3.60 (d, J = 17.1 Hz, IH), 3.54 (d, J= 17.1 Hz, I H), 3.24 (d, J = 17.2 Hz, I H), 2.48-2.36 (m, 4H), 2.17-2.21 (m, 2H), 2.04-2.1 1 (m, 2H), 1.95-1.83 (m, 2H), 1.62-1.51 (m, 2H), 1.49 (d, J = 6.4 Hz, 6H), 1.20-1.32 (m, 12H); 13C
53412 v2 'DC 132 NMR (CDCl3, 100 MHz) δ 168.02, 167.85, 159.08, 158.83, 157.23, 155.55, 155.36, 154.19, 140.75, 138.23, 138.19, 137.75, 136.93, 136.74, 132.32, 132.28, 128.34 (x 2), 128.31 (x 2), 128.18, 128.09 (x 2), 127.99 (x 2), 127.71, 127.63, 125.50, 1 18.82, 1 18.56, 1 18.34, 108.84, 108.50, 101.72, 101.68, 93.49, 93.44, 93.12 (x 2), 77.21, 76.02, 75.88, 71.18, 70.99, 64.47, 64.45, 64.33, 64.31, 39.99, 39.96, 34.87, 32.42, 32.31, 31.63, 31.09, 28.86, 20.25, 20.19, 15.04 (x 2), 14.98 (x 2); HRMS (ESI) m/z 560.2627 ([M+Naτ], C31H39NO7Na requires 560.2619.
EXAMPLE 19: General procedure for the synthesis of compounds 2-155. [00317] As depicted in Scheme 20, to a solution of compound 2-154 (1.0 equiv) in MeOH (0.02 M) was added PS-TsOH (10.0 equiv, 3.2 mmol/g) and the suspension was shaken at 40 0C for 4 h. The reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. The crude product obtained was submitted without further purification to the next step. Thus to a solution in CH2Cl2 (0.02 M ) of this crude at 23 0C were added DHP (1.0 equiv) and PS-TsOH (cat, 3.2 mmol/g) and the mixture was stirred for 5 hours. After that time the mixture was filtered, the solvents were evaporated under reduced pressure, and purification by PTLC (SiO2, 30% EtOAc/cyclohexane) afforded two different diastereoisomers 1 : 1 of 2-155 (-65%). An illustrative example of compound 2-155 follows; its characterization data are in the text below the figure. Illustrative Compound 2-155:
Figure imgf000134_0001
Less polar diastereoisomers: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 9.25 (s, IH), 9.24 (s, IH), 7.46-7.33 (m, 10H), 6.29 (s, IH), 6.26 (s, IH), 6.07-6.02 (m, 2H), 5.75 (d, J = 15.8 Hz, IH), 5.69 (d, J = 15.8 Hz, I H), 5.44-5.38 (m, 6H), 5.23 (s, 4H), 5.03 (d, J = 8.8 Hz, 2H), 4.34-4.13 (m, 6H), 3.69-3.63 (m, 2H), 2.70-2.67 (m, 2H), 2.30-2.16 (m, 6H), 2.08-1.94 (m, 8H), 1.73- 1.65 (m, 8H), 1.42 (t, J = 6.4 Hz, 3H), 1.39 (t, J - 7.0 Hz, 3H); HRMS (ESI) m/z 528.2562 ([M+Naτ], C30H35NO6Na requires 528.2357.
53412 v2< DC J 33 More polar diastereoisomers: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 11.61 (s, IH), 9.27 (s, I H), 7.41 -7.33 (m, 5H), 6.62 (d, J = 16.4 Hz, IH), 6.47 (s, I H), 6.15-6.07 (m, IH), 5.50-5.38 (m, 3H), 5.16 (s, 2H), 5.04 (d, J = 10.5 Hz, I H), 4.30 (d, J = 15.2 Hz, I H), 4.24 (d, J = 10.5 Hz, I H), 3.84 (d, J = 15.2 Hz, I H), 3.66 (t, J = 1 1.4 Hz, I H), 2.71 -2.65 (m, I H), 2.28-2.08 (m, 6H), 1 .73-1.64 (m, 5H), 1.38 (t, J = 7.0 Hz, 3H); HRMS (ESI) m/z 528.2494 ([M+Naτ], C30H35NO6Na requires 528.2357.
EXAMPLE 20; General procedure for the synthesis of compounds 2-128. [00318] As depicted in Scheme 21 , to a solution of pochonin D (2-85, X = Cl and R = Me) (25 mg, 71.2 μmol) in DMF (5 ml) TBSCl (53.6 mg, 356 μmol) and imidazole (23.6 mg, 356 μmol) were added and the mixture was stirred for 3 hours at room temperature. Purification by column chromatography (SiO2 0-30% EtOAc/cyclohexane gradient) afforded after evaporation 2-128 (40 mg, 98%). An illustrative example of compound 2-128 follows; its characterization data are in the text below the figure. Illustrative Compound 2-128:
Figure imgf000135_0001
1H NMR (CDCl3, 400 MHz, 25 0C) δ 6.71 (dt, J = 15.3, 7.3 Hz, IH), 6.45 (s, IH), 5.81 (d, J = 15.3 Hz, IH), 5.25 (s, 2H), 5.04-5.03 (m, IH), 3.89 (d, J= 17.4 Hz, IH), 3.57 (d, J= 17.4 Hz, I H), 2.31-2.04 (m, 6H), 1.35 (d, J= 6.4 Hz, 3H), 1.03 (s, 9H), 0.99 (s, 9H), 0.28-0.24 (m, 12H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 195.8, 166.8, 152.9, 151.7, 146.5, 132.7, 131.9, 128.6, 126.8, 122.8, 1 19.7, 1 10.7, 71.9, 45.6, 38.5, 30.9, 25.7 (x 4), 25.6 (x 4), 18.7, 18.3, - 4.1 (x 2), -4.4 (x 2) ; HRMS (ESI) m/z 601.2568 ([M+Na], C30H47ClO5Si2Na requires 601.2543).
EXAMPLE 21 : General procedure for the synthesis of compounds 2-157. [00319] As depicted in Scheme 21, to a solution of compound 2-128 (1.0 equiv) in pyridine/AcOH (5/1 , 250 μL) was added the corresponding hydroxylamine (5.0 equiv) and the mixture was heated up to 40 0C. After stirring overnight the solvents were evaporated under reduced pressure, and filtration on SiO2 with a mixture of 30% EtOAc/cyclohexane afforded after evaporation two isomers of 2-157 ~90%. An illustrative example of compound 2-157 follows; its characterization data are in the text below the figure.
53412 \2 DC 134 Illustrative Compound 2-157;
Figure imgf000136_0001
cis oxime 1H NMR (CDCl3, 400 MHz) δ 7.42 (bd, J = 6.4 Hz, 2H), 7.36 (bdd, J = 7.5, 6.9 Hz, 2H), 7.34-7.32 (m, I H), 6.52 (d, J = 16.1 Hz, I H), 6.38 (s, IH), 6.18-6.10 (m, I H), 5.36- 5.32 (m, 2H), 5.16 (bs, 2H), 4.99-4.95 (m, I H), 3.79-3.76 (m, 2H), 2.40-1.99 (m, 6H), 1.45 (d, J = 6.2 Hz, 3H), 1 .03 (s, 9H), 0.99 (s, 9H), 0.28 (s, 3H), 0.26 (s, 3H), 0.20 (s, 6H) ; trans oxime 1H NMR (CDCl3, 400 MHz) δ 7.44 (bd, J = 6.5 Hz, 2H), 7.37 (bdd, J = 7.6, 6.9 Hz, 2H), 7.33-7.31 (m, I H), 6.41 (s, I H), 6.04-5.97 (m, I H), 5.48 (bd, J = 15.0 Hz, IH), 5.29- 5.27 (m, I H), 5.22 (bs, 2H), 5.00-4.95 (m, IH), 3.98-3.89 (m, 2H), 2.39-2.02 (m, 6H), 1.37 (d, J = 5.9 Hz, 3H), 1.04 (s, 9H), 0.99 (s, 9H), 0.28 (s, 3H), 0.27 (s, 3H), 0.23 (s, 3H), 0.22 (s, 3H).
EXAMPLE 22: General procedure for the synthesis of compounds 2-158. [00320] As depicted in Scheme 21 , to a solution of corresponding compound 2-157 (1.0 equiv) in THF was added TBAF (2.5 equiv, I M solution in THF) and the mixture was stirred at room temperature for 2 hours. The solvents were then evaporated under reduced pressure, and filtration on SiOo with a mixture of 30% EtOAc/cyclohexane afforded after evaporation, compounds 2-158 in >85 % yield. An illustrative example of compound 2-158 follows; its characterization data are in the text below the figure. Illustrative Compound 2-158;
Figure imgf000136_0002
S3412 V2/DC 135 Cis: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 1 1.52 (s, IH), 7.45-7.34 (m, 5H), 6.64 (s, IH), 6.09-6.02 (m, 2H), 5.34-5.25 (m, 4H), 5.18-5.08 (m, 2H), 4.33 (d, J = 17.0 Hz, IH), 4.15 (d, J - 17.6 Hz, I H), 2.65-2.59 (m, IH), 2.27-2.14 (m, 3H), 2.04-2.00 (m, IH), 1.88-1.83 (m, IH), 1.30 (t, J = 6.4 Hz, 3H); HRMS (ESI) m/z 478.1372 ([M+Na+], C25H26ClNO5Na requires 478.1392).
Trans: 1H NMR (CDCl3, 400 MHz, 25 0C) δ 1 1.73 (s, IH), 7.32-7.26 (m, 5H), 6.64 (s, I H), 6.50 (d, J = 16.4 Hz, I H), 6.06-5.98 (m, 2H), 5.43-5.24 (m, 3H), 4.91 (s, 2H), 4.22 (s, 2H), 2.61-2.55 (m, I H), 2.46-2.33 (m, 2H), 2.20-2.02 (m, 3H), 0.98 (t, J = 6.4 Hz, 3H); HRMS (ESI) m/z 478.1522 ([M+Naτ], C25H26ClNO5Na requires 478.1392).
EXAMPLE 23: General procedure for the synthesis of compounds 2a- 1 via Mitsunobu cyclization.
[00321] As depicted in Scheme 24, the preparation of compound 2a-l begins with commercially available orcinol, 8. The description below is not intended to be limiting and alternate analogs may be prepared with the same process. Formylation of orcinol, synthesis of aldehyde 9. POCl3 (54.9 mL, 600 mmol, 2.0 equiv) was added slowly to a flask containing DMF (100 mL) at 0 0C. To this mixture was then added a solution of orcinol (42.65 g, 300 mmol, 1.0 equiv) as a solution in DMF (100 mL) and the reaction was allowed to warm up to 23 0C and stirred overnight. Then, the reaction was cooled to 0 0C and 200 mL of ice-water were added. The pH of the mixture was adjusted between 6 and 7 by addition of 10% of NaOH. The mixture was allowed to stand for 30 minutes and then the precipitates were filtered to afford the desired aldehyde 9 (29.2 g, 64%) as a white solid which was used in the subsequent step without further purification: Rf= 0.31 (Hexane/EtOAc 3/1); 1H NMR ((CD3)2CO, 400 MHz, 25 0C) δ 12.48 (s, IH), 10.10 (s, IH), 9.53 (s, I H), 6.30 (d, J = 2.4 Hz, IH), 6.17 (d, J = 2.0 Hz, IH), 2.54 (s, 3H); 13C NMR ((CD3)2CO, 100 MHz, 25 0C) δ 194.3, 167.3, 166.2, 146.1, 1 13.8, 1 1 1.5, 101.4, 18.2; HRMS (MALDI-TOF) m/z 175.0373 ([M+Na+], C8H8O3Na requires 175.0371).
Figure imgf000137_0001
53412 v2/ DC 136 Bis-EOM-protection, synthesis of aldehyde 9b. To a solution of bis-phenol 9 (29.2 g, 191.9 mmol) in DMF (400 mL) at 0 0C were sequentially added /Pr2NEt (95.2 mL, 575.7 mmol, 3.0 equiv) and EOMCl (52.3 mL, 575.7 mmol, 3.0 equiv). The reaction was allowed to warm up to 23 0C and stirred overnight. Then, the reaction was quenched with sat. NH4CUq (100 mL) and further diluted with CH2Cl2 (400 mL). The organic layer was separated and washed with sat. NH4Claq (100 mL x 2), brine (100 mL x 2), and dried over anhydrous Na2SO4 (5.0 g). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO?, Hexane/EtOAc 8/1 ) afforded protected aldehyde 9b as a colorless oil (46,5 g, 90%): Rf= 0.51 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 10.48 (s, I H), 6.71 (d, J = 2 Hz, IH), 6.49 (d, J = 2 Hz, I H), 5.27 (s, 2H), 5.22 (s, 2H), 3.72 (q, J= 6.9 Hz, 2H), 3.69 (q, J = 6.9 Hz, 2H), 2.54 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H), 1.19 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 190.6, 163.0, 162.1 , 144.2, 1 18.6, 1 12.3, 100.6, 93.6, 92.8, 64.8, 64.7, 22.2, 15.1 (x 2); HRMS (MALDI-TOF) m/z 291.1224 ([M+Naτ], C 14H20O5Na requires 291.1209).
Figure imgf000138_0001
Oxidation-chlorination, synthesis of acid 10. To a solution of aldehyde 9b (46.5 g, 173 mmol) in THF (200 mL) was added a solution Of NaH2PO4 (67.5 g, 433 mmol, 2.5 equiv) in H2O (200 mL) and the mixture was cooled down to 0 0C. Then a solution Of NaClO2 (48.9 g, 85%, 433 mmol, 2.5 equiv) in H2O (200 mL) was added slowly to the reaction. After stirring overnight the mixture was diluted with EtOAc (500 mL), washed with brine (200 mL x 3) and dried over Na2SO4 (5.0 g). Filtration and evaporation of the solvents under reduced pressure afforded acid 10 (49.6 g, 90%) as a white solid which was used in the next step without further purification: Rf= 0.66 (CH2Cl2MeOH 9/1); 1H NMR ((CD3)2CO, 400 MHz, 25 0C) δ 7.08 (s, I H), 5.34 (s, 2H), 5.25 (s, 2H), 3.75 (q, J = 7.1 Hz, 2H), 3.71 (q, J = 7.1 Hz, 2H), 2.34 (s, 3H), 1.19 (t, J = 7.1 Hz, 3H), 1.17 (t, J = 7.1 Hz, 3H); 13C NMR ((CD3)2CO, 100 MHz, 25 0C) 6 168.1, 154.9, 153.9, 135.1 , 121.8, 1 17.3, 102.8, 94.7, 94.6, 65.2, 65.0, 17.6, 15.4, 15.4; HRMS (MALDI-TOF) m/z 341.0713 ([M+Na~], Ci4Hi9ClO6Na requires 341.0768).
53412 v2/DC 137
Figure imgf000139_0001
10 11
Mitsunobu esterification, synthesis of ester 11. To a solution of acid 10 (23.5 g, 73.7 mmol) in toluene (300 mL) at 0 0C, were added sequentially trimethylsilylethanol (12.7 mL, 88.5 mmol, 1.2 equiv), PPh3 (28.9 g, 1 10.5 mmol, 1.25 equiv) and DIAD (21.7 mL, 1 10.5 mmol, 1.25 equiv). The reaction was allowed to warm up to 23 0C and stirred for 3 hours. Evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 20/1 and 10/1) afforded ester 11 (24.7 g, 80%) as a white solid: Rf= 0.60 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 6.98 (s, IH), 5.28 (s, 2H), 5.18 (s, 2H), 4.41 -4.36 (m, 2H), 3.75 (q, J - 6.9 Hz, 2H), 3.70 (q, J - 6.9 Hz, 2H), 2.32 (s, 3H), 1,21 (t, J = 7.1 Hz, 3H), 1 ,20 (t, J = 7.1 Hz, 3H), 1.12-1.07 (m, 2H), 0.06 (s, 3H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 167.7, 154.2, 153.1 , 135.0, 120.5, 1 17.3, 102.0, 94.0, 93.9, 64.7, 64.5, 63.8, 17.6, 15.1 (x 2), -1.41 (x 3); HRMS (MALDI-TOF) m/z 441.1487 ([M+Na+], C19H31ClO6SiNa requires 441.1476).
2Me LiAIH4, THF HQ
MeO5C
16 17a
Reduction of diester 16, synthesis of diol 17a. To the suspension Of LiAlH4 (15.2 g, 400 mmol, 4 equiv) in THF (300 mL) at 0 0C was added drop wise a solution of trans-3- hexenedioic acid dimethyl ester (17.2 g, 100 mmol) in THF (100 mL). The reaction was stirred for 2 hours. After which, the reaction was quenched by addition Of H2O (15.2 mL), 15% of NaOH (15.2 mL), and H2O again (45.6 mL). Then the mixture was poured into Et2O (150 mL), stirred for 30 minutes, and filtered over Na2SO4 (5 g). Evaporation of the solvents under reduced pressure afforded diol 17a which was used without further purification (1 1.0 g, 95%): Rf= 0.53 (CH2Cl2/Me0H 9/1 ); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 5.55-5.52 (m, 2H), 3.66 (dt, J = 5.6, 5.2 Hz, 2H), 2.33-2.28 (m, 4H), the OH is not visible; 13C NMR (CDCl3, 100 MHz, 25 0C) δ 129.8 (x 2), 62.0 (x 2), 36.16 (x 2); HRMS (MALDI-TOF) m/z 139.0734 ([M+Nal, C6Hi2O2Na requires 139.0735).
53412 v2/DC 138 TBDPSCI, NaH
^OTBDPS
17a 17
TBDPS-monoprotection of 17a, synthesis of alcohol 17. To a suspension of NaH (1.90 g, 47.4 mmol, 1.0 equiv) in THF (100 mL) at 0 0C a solution of diol 17a (5.5 g, 47.4 mmol, 1.0 equiv) in THF (20 mL) was added. After stirring for 45 minutes, TBDPSCl (12.4 ml, 47.4 mmol, 1.0 equiv) was added. The reaction mixture was allowed to warm up to 23 0C and stirred for 1 hour. Then, it was quenched with sat. NH4CIa4 (200 mL) and extracted with EtOAc (150 mL x 3), the combined organic layers were washed with brine (200 mL x 2) and dried over Na2SC>4 (1 Og). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 20/1, 10/1 and 5/1) afforded desired alcohol 17 (1 1.5 g, 68%) as a colorless oil: Rf= 0.46 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.68-7.65 (m, 4H), 7.43-7.36 (m, 6H), 5.56 (dt, J = 15.2, 6.4 Hz, IH), 5.44 (dt, J = 15.2, 6.8 Hz, I H), 3.69 (t, J = 6.4 Hz, 2H), 3.62 (t, J = 6.3 Hz, 2H), 2.31-2.24 (m, 4H), 1.05 (s, 9H), the OH is not visible; HRMS (MALDI-TOF) m/z 377.1927 ([M+Na+], C22H30O2SiNa requires 377.1913).
a. I2, PPh3, Imid. b. KCN
17 19
Synthesis of nitrile 19. To a solution of alcohol 17 (1 1.5 g, 32.4 mmol) in CH2Cl2 (150 mL) at O0C were added imidazole (4.30 g, 47.0 mmol, 1.45 equiv), triphenylphosphine (9.35 g, 35.7 mmol, 1.1 equi) and iodine (9.06 g, 35.7 mmol, 1.1 equiv) and the resulting solution was stirred at O0C for 6 hours. Then, the mixture was diluted with Et2O/Hexane 1/1 (300 mL), washed with sat. NaHCO3aq (150 mL x 2), sat. Na2S2O5aq (150 mL), brine (150 mL) and dried over Na2SO4 (5.0 g). The solvents were evaporated and resulting oily solids were triturated with hexanes (200 mL x 2). Filtration and evaporation of the hexanes afforded the corresponding alkyl iodide 18 as a colorless oil which was directly used in the next step without further purification. Rf= 0.46 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.70-7.67 (m, 4H), 7.45-7.36 (m, 6H), 5.58 (dt, J = 15.2, 6.8 Hz, I H), 5.44-5.37 (dt, J = 15.2, 6.4 Hz, IH), 3.69 (t, J = 6.4 Hz, 2H), 3.1 1 (t, J= 7.2 Hz, 2H), 2.54 (dt, J = 7.2, 6.8 Hz, 2H), 2.26 (dt, J = 6.4, 6.4 Hz, 2H), 1.05 (s, 9H); HRMS (MALDI-TOF) m/z 487.0923 ([M+H+], C22H29IOSiH requires 487.0930).
53412 v2/DC 139 [00322] To a solution of crude alkyl iodine 18 in DMSO (200 mL) was added KCN (21.0 g, 324 mmol, 1.0 equiv). The resulting mixture was stirred for 2 hours at 60 0C and then the reaction mixture was cooled down to 23 0C, diluted with EtOAc (200 mL), washed with water (200 mL), brine (100 mL), and dried over Na2SO4 (5.0 g). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 20/1 and 10/1 ) afforded desired nitrile 19 (10.6 g, 90%) as a colorless oil: Rf= 0.46 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.71-7.69 (m, 4H), 7.45-7.39 (m, 6H), 5.60 (dt, J= 15.2, 6.4 Hz, IH), 5.52-545 (m, IH), 3.73 (t, J= 6.4 Hz, 2H), 2.36-2.28 (m, 6H), 1.08 (s, 9H); 13C NMR (CDCl3, 100 MHz, 25 0C) 135.7 (x 4), 134.0 (x 2), 130.5, 129.7 (x 2), 127.8, 127.7 (x 4), 119.4, 63.6, 35.9, 28.6, 27.0 (x 3), 19.3, 17.6; HRMS (MALDI- TOF) m/z 386.1962 ([M+Na+], C23H29OSiNNa requires 386.1916).
Figure imgf000141_0001
21
Synthesis of ylide 21. To a solution of N-methoxy-N-methylhydroxylamine hydrochloride (48.52 g, 500 mmol, 1.0 equiv) in CH2Cl2 (1.0 L) at 00C were added pyridine (80.63 mL, 1.0 mol) and chloroacetic anhydride (85.14 g, 500 mmol, 1.0 equiv). The resulting mixture was stirred for 15 min at O0C, then warm up to 230C and stirred overnight. The reaction mixture was then poured carefully into sat. NaHCO33Q solution (1.0 L) and stirred 1 hour, after which the layers were separated, the aqueous phase was extracted with CH2Cl2 (400 mL) and the combined organic layers were washed with IN HCl (200 mL x 2), brine (200 mL x 2), dried over Na2SO4 (10 g) and filtered. Evaporation of the solvents under reduced pressure afforded the corresponding acetamide (N-methoxy-N-methyl acetamide-2-chloride) as a green oil which was used in the next step without further purification. To a solution of this acetamide in CH3CN (800 mL) was added Ph3P (107.98 g, 41 1.7 mmol, 0.82 equiv) and the resulting mixture was refluxed for 18 hours. Then the solvents were removed under vacuum and the resulting viscous oil was dissolved in CH2Cl2 (1.0 L), washed with 2N KOH (400 mL x 2), brine (400 mL) and dried over Na2SO4 ( 10.0 g). Filtration and evaporation of the solvents under reduced pressure afforded ylide 21 as a thick oil which solidified by standing (146.5 g, 80% over two steps). This compound was used in the next step without further purification. Rf= 0.85 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) 5 7.71-7.65 (m, 6H), 7.55-7.50 (m, 3H), 7.48-7.42 (m, 6H), 3.74 (s, 3H), 3.08 (s, 3H), 1.86 (s, IH); 13C NMR
53412 v2 DC |4Q (CDCl3, 100 MHz, 25 0C) 133.3 (x 3), 133,2 (x 3), 131.9 (x 3), 128.9 (x 3), 128.8 (x 3), 127.9, 61.3, 35.9. OTBDPS
Figure imgf000142_0001
Synthesis of Weinreb amide 12. To a solution of nitrile 19 (22.94 g, 63.0 mmol, 1.0 equiv) in CH2Cl2 (250 mL) at -78 0C, DIBAL (66.3 mL, IM in toluene, 1.05 equiv) was added and the reaction was stirred for 30 min. To quench the reaction, a solution of sat. K/Na(tartrate)aq (300 mL) was added and the biphasic mixture was stirred until it became a clear biphasic system (over 2 h). The two phases were separated and the aqueous phase was further extracted with CH2Cb (200 mL x 3). The combined organic layers were then washed with brine (200 mL) and dried over Na2SO4 (1Og). Filtration followed by evaporation of the solvents under reduced pressure afforded the corresponding aldehyde 20 which was used in the next step without further purification. Thus, the crude aldehyde 20 was dissolved in CH2Cl2 (200 mL) at 23 0C and ylide 21 was added (30 g, 81.9 mmol, 1.3 equiv). The reaction was then stirred overnight. Evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 20/1, 10/1 and 3/1) afforded desired Weinreb amide 12 (14.5 g, 61 % over two steps) as a colorless oil: Rf= 0.30 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.69-7.67 (m, 4H), 7.43-7.36 (m, 6H), 6.98 (dt, J = 15.3, 7.1 Hz, IH), 6.40 (d, J = 15.3 Hz, IH), 5.48-5.46 (m, 2H), 3.68 (t, J = 6.6 Hz, 2H), 3.67 (s, 3H), 3.24 (s, 3H), 2.32-2.24 (m, 4H), 2.19-2.14 (m, 2H), 1.06 (s, 9H); 13C NMR (CDCl3, 100 MHz, 25 0C) 167.1 , 147.2, 135.7 (x 4), 134.1 , 131.0 (x 2), 129.6 (x 2), 127.8 (x 4), 127.7, 1 19.0, 64.0, 61.7, 36.1 , 32.6, 32.5, 31.5, 27.0 (x 3), 19.3; HRMS (MALDI-TOF) m/z 474.2432 ([M+Na~], C27H37NO3SiNa requires 474.2440).
a. LDA
Figure imgf000142_0002
11 13
53412 v2.'DC 141 Coupling of toluate 11 and Weinreb amide 12, synthesis of ketone 13. A solution of compound 11 (8.38 g, 20.0 mmol, 1.0 equiv) in anhydrous THF (120 mL) at -78 0C was treated with freshly prepared LDA (71.4 mL, 0.56 M, 40.0 mmol, 2.0 equiv) added via cannula. Immediately after, a solution of Weinreb amide 12 (8.13 g, 18 mmol, 0.9 equiv) in THF (15 mL) at -78 0C was added via cannula. The resulting mixture was then stirred for 15 minutes and the reaction was quenched by addition of sat. NH4ClnC1 (20 mL), Upon warming to 23 0C, the reaction mixture was extracted with EtOAc (200 mL x 2), and the combined organic layers were then washed with brine (100 mL) and dried over Na2SO4 (5.0 g). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 20/1, 10/1 and 5/1) afforded desired ketone 13 (20.7 g, 65%): Rf= 0.48 (Hexane/EtOAc 3/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.70-7.68 (m, 4H), 7.42-7.38 (m, 6H), 7.10 (s, I H), 6.94 (dt, J= 15.8, 6.7 Hz, IH), 6.19 (d, J = 15.8 Hz, I H), 5.49-5.46 (m, 2H), 5.30 (s, 2H), 5.22 (s, 2H), 4.36-4.32 (m, 2H), 4.06 (s, 2H), 3.79-3.68 (m, 6H), 2.30-2.25 (m, 4H), 2.18-2.13 (m, 2H), 1.23 (t x 2, J = 7.1 Hz, 6H), 1.07 (s, 9H), 1.08- 1.03 (m, 2H), 0,06 (s, 9H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 194.5, 167.0, 154.6, 153.9, 147.1 , 135.5 (x 4), 133.9 (x 2), 132.8, 130.5, 129.5 (x 2), 129.1 , 127.9, 127.6 (x 4), 120.3, 1 17.7, 103.1 , 93.9, 93.8, 64.6, 64.4, 63.8, 63.6, 60.3, 59.9, 43.0, 35.9, 32.4, 31.1, 26.8 (x 3), 22.6, 22.0, 19.2, 17.4, 15.0, 14.2, 14.1, -1.40 (x 3); HRMS (MALDI-TOF) m/z 831.3380 ([M+Naτ], C44H6 IClO8Si2Na requires 831.3491).
Figure imgf000143_0001
Synthesis of oximes 14a. To a solution of ketone 13 (18,6 g, 25.5 mmol) in pyridine (50 mL) at 40 0C, carboxymethoxylamine hemihydrochloride (13.98 g, 127.5 mmol) was added and the reaction was stirred at such temperature for 24 hours. After evaporation of the pyridine, the residues were dissolved in CH2Cl2 (200 mL) and washed with sat. NH4Claq (50 mL x 2), brine (50 mL x 2) and dried over anhydrous Na2SO4 (2.0 g). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 5/1 and 1/1 ) afforded desired oximes 14a (10.1 g, 50%) as a mixture of E/Z isomers in a 1/1 ratio: Rf= 0.51 (CH2Cl2MeOH 19/1); 1H NMR (CDCl3, 400 MHz, 25 0C) 8 7.69-7.65 (m, 8H), 7.42-7.35 (m,12H), 7.07 (s, IH), 7.05 (s, IH), 6.74 (d, J = 15.8 Hz, IH), 6.36 (dt, J = 15.8,
53412 v2/DC 142 6.8 Hz, I H), 6.07 (dt, J = 15.8, 6.8 Hz, I H), 5.79 (d, J = 15.8 Hz, IH), 5.48-5.43 (m, 2H), 5.39-5.34 (m, 2H), 5.30 (s, 2H), 5.28 (s, 2H), 5.21 (s, 2H), 5.19 (s, 2H), 4.63 (s, 2H), 4.46 (s, 2H), 4.35-4.29 (m. 4H), 4.03 (s, 2H), 3.88 (s, 2H), 3.80-3.60 (m, 12H), 2.31-2.25 (m, 6H), 2.17-2.15 (m, 2H). 2.10-2.06 (m, 2H), 2.00-1.97 (m, 2H), 1.27-1.21 (m, 12H), 1.08 (s, 9H), 1.07 (s, 9H), 1.07-1.05 (m, 4H), 0.09 (s, 9H), 0.08 (s, 9H), the OH from the acids are not visible; 13C NMR (CDCl3, 100 MHz, 25 0C) 5 172.0, 171.9, 167.9, 167.3, 157.0, 155.6, 154.6, 154.5, 153.7, 153.7, 141.4, 137.8, 135.7 (x 8), 134.6, 134.1 (x 4), 133.7, 131.2, 130.8, 129.7 (x 4), 127.9, 127.7 (x 8), 127.5, 124.2 (x 2), 120.4, 1 19.1, 1 17.5, 1 17.4, 103.2, 102.8, 94.1 , 94.0, 93.9, 93.8, 71.1 , 70.5, 64.9, 64.9, 64.7, 64.6, 64.5, 64.0, 64.0, 63.9, 36.1 (x 2), 33.5, 33.1 , 32.9, 31.9, 31.9, 29.4, 27.0 (x 6), 19.3 (x 2), 17.5, 17.3, 15.1 (x 4), -1.4 (x 6).
Figure imgf000144_0001
Synthesis of amides 14. To the solution of acid 14a (10.0 g, 1 1.33 mmol, 1.0 equiv) in CH2Cl2 (60 mL) at 0 0C, was added EDCHCl (2.6 g, 13.6 mmol, 1.2 equiv) followed by a solution of piperidine (1.34 mL, 13.6 mmol, 1.2 equiv) in CH2Cl2 (10 mL). The reaction was allowed to warm up to 23 0C , and stirred for 3 hours. The reaction mixture was then diluted with CH2Cl2 (200 mL), washed with sat. NH4Cldq (100 mL), brine (100 mL), and dried over anhydrous Na2SO4 (5 g). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO?, Hexane/EtOAc 2/1 and 1/1) afforded desired amides 14 (6.60 g, 63%) as a mixture of two isomers in a 1/1 ratio: Rf= 0.31 (Hexane/EtOAc 3/1 ); 1H NMR (CDCU, 400 MHz, 25 0C) δ 7.68-7.64 (m, 8H), 7.41-7.33 (m,12H), 7.04 (s x 2, 2H), 6.71 (d, J= 16.0 Hz, I H), 6.21 (dt, J = 16.0, 6.7 Hz, IH), 6.07 (dt, J = 16.0, 6.7 Hz, I H), 5.73 (d, J = 16.0 Hz, I H), 5.45-5.39 (m, 2H), 5.37-5.32 (m, 2H), 5.28 (s, 2H), 5.25 (s, 2H), 5.19 (s, 4H), 4.73 (s, 2H), 4.60 (s, 2H), 4.37-4.27 (m, 4H), 3.95 (s, 2H), 3.87 (s, 2H), 3.80-3.60 (m, 12H), 3.55-3.52 (m, 2H), 3.51 -3.48 (m. 2H), 3.38-3.35 (m, 2H), 3.32-3.29 (m, 2H), 2.26-2.16 (m, 6H), 2.09-2.06 (m, 2H), 2.01-1.98 (m, 2H), 1.95-1.91 (m, 2H), 1.61 -1.46 (m, 12H), 1.28-1.17 (m, 12H), 1.05-1.01 (m, 4H), 1.04 (s, 9H), 1.03 (s, 9H), 0.05 (s, 9H), 0.04 (s, 9H); πC NMR (CDCl3, 100 MHz, 25 0C) δ 167.1 , 167.0 (x 2), 166.8, 155.6, 154.3, 154.2, 153.5, 153.4, 153.1 , 139.2, 136.5, 135.6 (x 8), 134.7, 134,6, 134.1 (x 4), 131.4, 131.1,
53412 v2/DC 143 129.6 (x 4), 127.6 (x 8), 127.6, 127.1 , 124.1 , 121.0, 120.9, 1 19.5, 1 17.9, 1 17.7, 103.1, 102.9,
94.1 , 94.0, 93.9, 73.1 , 73.0, 64.8, 64.7, 64.6, 64.5, 64.0, 63.9, 63.9, 63.6, 60.4, 53.5, 46.4,
46.2, 43.0, 42.9, 36.1 , 33.4, 33.0, 32.9, 32.0, 31.9, 30.0, 26.9 (x 6), 26.6, 26.5, 25.6, 24.6, 19.3 (x 2), 17.4, 17.3, 15.1 (x 6), - 1.42 (x 6).
Figure imgf000145_0001
Silyl deprotection, synthesis of macrocyclization precursor 14b. To a solution of ester 14 (6.60 g, 6.9 mmol, 1.0 equiv) in THF (70 mL) at 23 0C TBAF (17.3 mL, IM in THF, 2.5 equiv) was added dropwise and the resulting mixture was stirred for 3 hours. Then, EtOAc (100 mL) was added to the reaction, and the resulting mixture was washed with IM HCl (60 mL x 3), brine (60 mL), and dried over Na2SO4 (1.0 g). Filtration and evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, EtOAc and EtOAc/MeOH 4/1) afforded desired acids 14b (3.78 g, 89%) as a mixture of two isomers in a 1/1 proportion: Rf= 0.35 (CH2CVMeOH 9/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.03 (s, I H), 6.99 (s, I H), 6.62 (d, J- 16.1 Hz, I H), 6.23 (dt, J = 16.1 , 6.9 Hz, IH), 6.1 1 (dt, J = 16.1 , 6.9 Hz, I H), 5.95 (d, J = 16.1 Hz, I H), 5.55-5.30 (m, 4H), 5.26 (s x 2, 4H), 5.21 (s, 2H), 5.20 (s, 2H), 4.72 (s, 2H), 4.63 (s, 2H), 4.05 (s, 2H), 4.00 (s, 2H), 3.81-3.66 (m, 8H), 3.65-3.57 (m, 4H), 3.54 (t, J = 5.4 Hz, 2H), 3.44 (t, J = 5.4 Hz, 2H), 3.31 (t, J = 5.4 Hz, 2H), 3.24 (t, J= 5.4 Hz, 2H), 2.23 (dt, J = 6.5, 6.5 Hz, 4H), 2.18-2.04 (m, 8H), 1.67-1.49 (m, 10H), 1.42-1.40 (m, 2H), 1.25-1.16 (m, 12H), OH from the alcohols and the acids are not visible; 13C NMR (CDCl3, 100 MHz, 25 0C) δ 168.73, 168.03, 156.47, 154.19, 152.99, 152.94, 140.23, 136.77, 134.37, 134.18, 132.45, 132.1 1 , 127.53, 127.23, 125.91, 122.46, 1 19.28, 1 16.76, 102.91 , 102.80, 94.16, 94.02, 93.96, 71.51 , 71.25, 64.92, 64.82, 64.54, 64.46, 62.1 1, 46.24, 45.60, 43.39, 43.27, 36.06, 35.98, 33.08, 32.80, 32.09, 31.86, 31.61, 29.20, 26.26, 26.18, 25.38, 25.30, 24.41 , 24.30, 15.28, 15.16; HRMS (MALDI-TOF) m/z 633.2562 ([M+Na+] C30H43ClN2O9Na requires 633.2555).
53412 v2/DC 144
Figure imgf000146_0001
Macrocyclization of 14b, synthesis of compounds 15. To a solution of acid 14b (3.78 g, 6.18 mmol) in toluene (200 mL) at 0 0C, was added PPh3 (2.43 g, 9.27 mmol) followed by a slow addition of DIAD (1.83 mL, 9.3 mmol). The reaction was allowed to warm to 23 0C and stirred for 5 hours. Evaporation of the solvents under reduced pressure followed by flash chromatography (SiO2, Hexane/EtOAc 4/1 and 1/1) afforded desired macrocycles 15 (2.09 g, 60%) as a mixture of two isomers in a 1/1 proportion: Rf= 0.50 (Hexane/EtOAc 1/1); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 7.05 (s, IH), 7.03 (s, IH), 6.51 (d, J = 16.0 Hz, IH), 6.16 (dt, J = 15.8, 6.5 Hz, IH), 6.00 (dt, J = 15.8, 7.7 Hz, IH), 5.44 (d, J= 16.0 Hz, IH), 5.32 (m, 4H), 5.28 (s, 2H), 5.26 (s, 2H), 5.21 (s, 2H), 5.20 (s, 2H), 4.80 (s, 2H), 4.71 (s, 2H), 4.23 (t, J = 5.1 Hz, 2H) , 4.18 (t, J= 5.1 Hz, 2H), 3.91 (bs, 2H), 3.80-3.66 (m, 10H), 3.60-3.51 (m, 4H), 3.48-3.46 (m, 2H), 3.41 -3.38 (m, 2H), 2.40-2.32 (m, 4H), 2.19-2.16 (m, I H), 2.1 1-2.00 (m, 7H), 1.67-1.51 (m, 12H), 1.23-1.19 (12H); πC NMR (CDCl3, 100 MHz, 25 0C) δ 167.5, 166.9, 154.8, 154.6, 154.4, 153.2, 141.0, 136.6, 134.1 , 132.6, 128.5, 127.6; 124.6, 121.6, 1 19.5, 1 17.5, 102.94 (x 2), 94.1 (x 2), 93.8 (x 2), 73.4, 73.2, 72.8, 72.7, 64.9, 64.8, 64.7 (x 2), 64.3 (x 2), 46.5, 46.3, 43.1 (x 2), 35.7, 35.6, 32.5, 32.4, 32.2, 32.1 , 31.9, 31.7, 30.2 (x 2), 26.7 (x 2), 26.6 (x 2). 25.7 (x 2), 24.7 (x 4), 15.2 (x 2), 15.1 (x 2); HRMS (MALDI-TOF) m/z 615.2438 ([M+Na"], CwH4iClN2O8Na requires 615.2450).
Figure imgf000146_0002
15 E/Z 2a-1 E = 2a-1
^3412 v2 DC 145 [00323] Deprotection, synthesis of 2a- 1 as an E/Z mixture, isolation of the E isomer and isomerization of the Z isomer. To a solution of macro cycles 15 (2.8 g, 4.72 mmol) in MeOH (60 mL) at 40 0C sulfonic acid resin (7.87 g, 3.0 mmol/g, 23.6 mmol) was added, and the suspension was stirred for 2 hours. The mixture was diluted with CH2Cl? (60 mL), filtered, and the resin was once rinsed with CH2CI2 (20 mL x 2). After removal of the solvent, the residue was re-dissolved in MeOH (50 mL) and sonicated. A precipitate began to form and the solution was allowed to stand for 12 h. The solution was filtered and rinsed with MeOH (30 mL). Evaporation of the combined solutions afforded 2a-l (1.13 g, 9: 1 E:Z as judged by LCMS). The remaining solid (pure Z-isomer, 900 mg) was dissolved in CH2Cl2 (180 mL) and treated with TFA (1.4 mL, 18.8 mmol) at 23 0C. The mixture was stirred for 12 hours, after which toluene (50 mL) was added and the solvents were evaporated (LCMS of this crude indicated a 1 : 1 mixture of E/Z isomers). The residue obtained was re-dissolved in CH2Cl? (30 mL). The insoluble precipitated was filtered and rinsed with MeOH (20 mL). Evaporation of the combined solutions afforded more of compound 2a-l (450 mg) again as a 9: 1 mixture in favor of the desired E isomer. The remaining solid (Z-isomer, 450 mg) was submitted to same isomerization conditions two more times. The combined batches of 8 were then purified by flash chromatography (70 g of C 18, CH3CN/H2O 35/65 and 0.01 % TFA, 50 mL/ min) to afford the pure E isomer as a white powder (1.10 g, 51% overall yield); Rf= 0.44 (Hexane/EtOAc 1/2); 1H NMR (CDCl3, 400 MHz, 25 0C) δ 1 1.64 (s, IH), 6.64 (s, I H), 6.01 (td, J = 15.5 7.5 Hz, I H), 5.1 1 (d, J= 15.5 Hz, I H), 5.10-5.03 (m, 2H), 4.85 (s, 2H), 4.37 (t, J = 4.8 Hz, 2H), 4.17 (s, 2H), 3.60 (t, J = 5.0 Hz, 2H), 3.46 (t, J = 5.0 Hz, 2H), 2.34 (q, J = 5.4 Hz, 2H), 2.10-2.02 (m, 2H), 1 .99-1.92 (m, 2H), 1.70-1.54 (m, 6H); 13C NMR (CDCl3, 100 MHz, 25 0C) δ 170.27, 167.42, 163.21 , 157.38, 155.18, 138.21, 135.62, 131.82, 129.13, 124.76, 1 15.55, 107.60, 103.47, 72.63, 65.03, 46.38, 43.31 , 33.21 , 32.76, 31.94, 31.84, 26.65, 25.64, 24.57; HRMS (MALDI-TOF) m/z 499.1638 ([M+H+], C24H29ClN2O6H requires 499.1612).
Biological Assays
[00324] The present invention includes assaying compounds of the present invention, i.e., test compounds, for use as therapeutics for NF2 or NFl . Specifically, three compounds, namely NXD30001 , NXD30002, and NXD3OOO3, are described below as representative compounds of the present invention. It is noted that NXD30001 is also referred to as E-2al in Table 1 of this application, NXD30002 is also referred to as Z-2-al in Table 1 of this application, and NXD30017 is also referred to as 2-al3 in Table 1 of this application. NF2-
53412 vZ'DC | 45 deficient or NFl -deficient cells lines as well as NF2-deficient cells or NFl -deficient cells obtained from a patient with NF2 or NFl , respectively, are treated with varying amounts of test compounds, and the effects of the test compound on degradation of one or more client proteins of HSP90, on HSP70 upregulation, and on activity or phosphorylation status of proteins in pathways associated with one or more client proteins of HSP90 are measured. Compounds that cause a decrease in the amount and/or activity, e.g. decrease or increase in phosphorylation, of one or more client proteins of HSP90, an upregulation of HSP70, or a decrease in activity, e.g. decrease or increase in phosphorylation, of proteins in pathways associated with one or more client proteins of HSP90 can be identified as a putative therapeutic for NF2 or NF 1.
[00325] The client proteins of HSP90 (e.g. ErbB2, Akt, c-Raf, Cdk4, and etc) are involved in many signaling pathways that control diverse cellular functions such as cell growth and proliferation, differentiation, and apoptosis. In order to determine proteins degraded and downstream pathways blocked by a HSP90 inhibitor in Nf2-/- Schwann cells or NfI-/- Schwann cells, one or more client proteins and downstream proteins such as ErbB2, Akt, c-Raf, Cdk4, mTOR, S6K, S6, GSK3, Mek, Erkl/2, Cyclin D can be monitored. For example, the effect of a HSP90 inhibitor on ErbB2 is assessed by comparing the amounts of ErbB2, phospho-ErbB2, and several phosphorylated downstream proteins (e.g. phospho-Akt, phospho-S6K, phospho-S6, and phospho-GSK3) in cells treated with the compound and those treated with vehicle (control).
[00326] Cells that are useful for treatment include, but are not limited to, NF2-deficient Schwann cells {e.g. HEI-193), NF2-deficient malignant mesothelioma cells (e.g. BAR and RAV), NF2-deficient meningioma cells {e.g. SF 1335), Nf2-deficient mouse cells (e.g. Schwann cells and mouse embryonic fibroblasts), and NF2-deficient schwannoma cells derived from schwannomas in a patient. For instance, HSP90 inhibitors can be evaluated in a panel of NF2 mutant human and mouse cells and cell lines. Antibodies to one or more client proteins of HSP90 (e.g. ErbB2, Akt, c-Raf, Cdk4, etc.) and activation-specific phospho- antibodies against these proteins or downstream proteins are ideal tools for this purpose (Cell Signaling Technology). [00327] Cells that are useful for treatment include, but are not limited to, NFl -deficient human MPNST cells {e.g. ST88-14, 88-3, 90-8, and sNF96.2) (Basu et al., 1992, Nature 356: 713-715; DeClue et al., 1992, Cell 69: 265-273; Wallace et al., 2000, Genes Chromosomes Cancer 27(2): 1 17-123; Muir et al., 2001 , Am. J. Pathol. 158: 501-513), primary neurofibroma cells derived from NFl patients, mouse /V/7/pJ.?-deficient MPNST cell lines
53412 v2 'DC | 47 established from cisNfl;p53 mice (Vogel et al., 1999, Science 286: 2176-2179), and NfI-/- mouse cells (e.g. Schwann cells, mouse embryonic cells, and leukemia cells).
1. NXD30001 downregulates Akt, ErbB2, and c-Raf in NF2-defϊcient mouse and human tumor cells
[00328] It has been shown that inhibition of HSP90 function in breast cancer cells causes down-regulation of Akt (Basso et al., 2002, J. Biol. Chem. 277(42): 39858-39866). The inventors of the present invention have determined that phospho-Akt levels are elevated in Np-/- mouse Schwann cells, NF2 defient human schwannomas, NF2-deficient meningioma xenografts, NF2-deficient mesothelioma xenografts as compared to wild-type mouse Schwann cells and/or normal human peripheral nerves (reference is made to PCT/US2007/70366 application entitled "Treatment of Neurofibromatosis with Inhibitors of a Signal Transduction Pathway," filed on June 4, 2007, which is herein incorporated by reference in its entirety). To assess the effect of HSP90 inhibitors on phospho-Akt in NF2 deficient cells, Western blots were prepared from Np-/- mouse Schwann cells as an example. The cells were treated with 0.02μM, 0.1 μM, 0.5μM NXD30001 for 24 hours. As control the same cells were treated with vehicle DMSO for 24 hours. The results show that treatment of NF2-deficient cells with NXD30001 caused a decline of phospho-Akt levels (Figures I A and I B). The loss of phospho-Akt was both dose- and time-dependent. [00329] To assess the effect of the compounds of the present invention on ErbB2 and c-Raf in NF2-deficient cells, Western blots were prepared from various NF2-deficient cells, including NfI-/- mouse Schwann cells, NF2-deficient meningioma cells, and NF2-deficient mesothelioma cells. The cells were treated similarly as described above. The inventors of the present invention have determined that treatment of NF2-deficient cells with NXD30001 caused a decline of both ErbB2 and c-Raf levels (Figure I A). The loss of ErbB2 and Raf proteins were both dose- and time-dependent, as shown in the result of Np-/- mouse Schwann cells treated with 0.1 μM NXD30001 for 3, 6, 12, 24 hours (Figure I B). The rapid decline of Akt phosphorylation might partially be caused by the degradation of ErbB2 upon NXD30001 treatment (Figure IA). ErbB2, one of the well-documented client proteins of HSP90, lies upstream of the Akt pathway in Schwann cells. Taken together, these data suggest that pharmacological inhibition of HSP90 function blocks the deregulated Akt pathway in NF2 mutant cells.
2. NXD30001 causes upregulation ofHSP70 in NF2-defιcient mouse and human tumor cells
53412 v2'DC 148 [00330] To assess the effect of the compouds of the present invention on HSP70 in NF2- deficient cells, Western blots were prepared from various NF2-deficient cells, including Nf2- /- mouse Schwann cells, NF2-deficient schwannoma cells, NF2-deficient meningioma cells, and NF2-deficient mesothelioma cells. The blot was prepared as described above and probed with anti-HSP70 antibody. The inventors of the present invention have found that treatment of NF2-deficient cells with NXD30001 caused an upregulation of HSP70 (Figure IA). The increase of HSP70 protein level was dose-dependent.
3. NXD30001 downregulates EGFR, At, c-Raf, and Cdk4 in NF 1 -deficient human MPNST cells
[00331] Ras-Raf-Mek-Erk 1/2 pathway is activated due to the loss of NFl in these cells. In addition, EGFR is overexpressed in a number of NFl -deficient human and mouse cells. Human malignant peripheral nerve sheath tumor (MPNST) cell ST88-14 was treated with increasing concentration (8-1000 nM) of NXD30001 for 24 hr. Cells were lysed and clarified lysates were subjected SDS-PAGE, followed by immunoblot analysis using indicated antibodies. Figure 2 demonstrates NXD30001 induced degradation of HSP90 client proteins EGFR, Akt, and c-Raf, as well as a cell-cycle regulating kinase Cdk4 with an IC50 of approximate 100 nM after 24 hr treatment. Equal lysate protein loading is shown by actin immunoblot.
4. NXDM)OOl, NXD30002, and NXD30017 inhibit proliferation of NFl- and NF2-deficient human and mouse cells
[00332] Inhibitors of HSP90 function is assayed for the ability to decrease the number of NF2 deficient tumor cells in a tumor, i.e., shrink a tumor, or reduce the proliferation of NF2 deficient tumor cells. The ability of inhibitors to decrease the number of NF2 deficient tumor cells in a sample or reduce proliferation of NF2 deficient tumor cells is assessed using a variety of methods known in the art. A small library of compounds of the present invention (total 14 oxime compounds) were assayed for inhibition of proliferation of NFl - and NF2- deficient cells. Cells used are Nfi-/- mouse embryonic stem cell (MESC), NF2-deficient human SF1335 meningioma cells, NF2-deficient HEI193 schwanoma cells, NF2-deficient BAR and RAV mesothelioma cells, NF2-plus BLA mesothelioma cells, and NFl -deficient human malignant peripheral nerve sheath tumor (MPNST) cells. Appropriate number of cells that can reach -70% confluence in 3 days (2000-6000 cells/well) were plated in 96-well plates. Various concentrations of each compound were added to the growing media and the
53412 v2OC 149 cells were then cultured for 3 days. A positive control compound (17-AAG) and a vehicle (DMSO) control were included with each assay. Upon completion of the incubation, media were gently removed and 100 μl of ATPlite solution (Perkin Elmer) was added in each well. Viable cells were measured by detecting luminescence generated from reaction of ATPlite solution and the ATP in the cells. IC50, the concentration needed for 50% inhibition of cell proliferation, were determined and analyzed for each compound using XLfit 4.1 software. Efficacious compounds preferably have about an IC50 < lOμM. IC50s of these compounds inhibiting proliferation of various NFl and NF2 cells are shown in Figure 3.
5. Pharmacodynamic assays measuring in vivo target inhibition
[00333] Various pharmacodynamic (PD) assays are useful to measure in vivo target inhibition.
[00334] The present invention also includes screening for degradation of one or more client proteins of HSP90 or upregulation of HSP70 using peripheral blood mononuclear cells ("PMBC PD assay"). In this assay, whole blood from groups of about 5 similarly treated normal and/or mice harboring NF2-deficient or NFl -deficient tumors at efficacious doses is collected and pooled in a single heparinized Vacutainer tube (BD biosciences, NJ), and peripheral blood mononuclear cells (PBMCs) are isolated as reported (Graff et ai, 2005, Cancer Res. 65(16): 7462-7469; Peralba et al, 2003, Clin. Cancer Res. 9(8): 2887-2892). PBMCs are collected at 0, 2, 4, 8, and 24 hours post dosing. PBMCs are lysed directly for Western blots or for immunoprecipitation first and then Western blots to detect target proteins. For example, HSP70 upregulation by a HSP90 inhibitor is detected in PBMCs (Ramanathan et al., 2007, Clin. Cancer Res. 13(6): 1769-1774). [00335] In yet another assay ("'tumor tissue PD assay"), either target protein degradation (e.g. ErbB2, c-Raf, Akt, Cdk4), phosphorylation, and HSP70 upregulation in tumor lysates by Western blots or in tumor sections (i.e. paraffin-embedded sections) by immunohistochemistry are used to assay the effects of the test compound on target proteins. Tumor tissues are harvested at the same time points as described above for PBMCs. Each tumor sample is divided in two pieces with one flash frozen in liquid nitrogen immediately and the other fixed in 10% buffered formalin and then paraffin-embedded. ErbB2, phospho- Akt, and phospho-S6 levels in Np -/-Schwann cell xenografts have been shown to be veiy high and can be readily detected by immunohistochemistry (reference is made to a PCT application entitled 'Treatment of Neurofibromatosis with Inhibitors of a Signal Transduction Pathway" referenced as Attorney Docket No. NEXG-005-01 WO, filed on June 4, 2007).
53412 v2/DC J 50 Phospho-Sό level in NfI-/- tumor cells is also elevated and is readily detected by immunohistochemistry (Johannessen et al., Proc. Natl Acad, Sci. U.S.A. 2005, 102(24):8573- 8578; Dasgupta et al., Cancer Res. 2005, 65(7):2755-2760).
6. Xenografts, Animal models, and Treatment
[00336] The ability of a test compound to act as a therapeutic in NF2 or NFl by modulating the HSP90 complex can also be assessed using animal xenograft experiments. Xenograft experiments are performed with subcutaneous tumor formation in nude mice or SCID mice using a human NF2-deficient malignant mesothelioma cell line RAV, a meningioma cell line or Nf2-/- SCs. A dermal neurofibroma (DNF) xenograft model has been established to evaluate compounds useful for treating NFl neurofibromas (PCT publication WO 06/083979). In particular, these experiments have allowed the inventors of the present invention to define the treatment starting point for each xenograft model. [00337] In order to assess the action of a test compound on the growth of the xenografts, tumor-bearing mice are randomized among control and treated groups with 8- 12 mice in each experimental group. For oral dosing, compounds are suspended in 0.5% (w/v) methyl cellulose or other suitable dosing vehicles known in the art. Dosing starts from a predefined starting point for each cell system (i.e. 100-200 mm3 at 3-5 days for Nf2-/- Schwann cells). Mice are dosed once or twice a day with high, medium, low doses and vehicle control for 4 weeks or less determined by preset tumor volume criteria. The actual dose should be calculated based on data of in vitro analyses, pharmacokinetic studies, and maximal tolerated dose (MTD) determined using normal mice or tumor-bearing mice. In some cases, it may be optimal to dose via intravenous, intraperitoneal or intralesional routes. For intraperitoneal dosing, mice are dosed twice daily, once daily, three times a week, two times a week, once a week. In case of significant variations in body weight during the treatment period, doses can be adjusted accordingly.
[00338] Mice are monitored daily and tumor growth, assessed based on tumor volume (V= [length x width"] π/6), and measured twice a week with calipers. Tumor formation is monitored for the duration of the treatment or longer, for instance, 3-6 weeks. Animals are sacrificed earlier when they meet predetermined criteria established for minimizing pain and suffering. Treatment efficacy endpoints are assessed in terms of the compound's effects on tumor growth of treated mice relative to that of control vehicle-treated mice. Two evaluation criteria are used in parallel: (/) Growth Inhibition, calculated as the ratio of the mean tumor volume of drug-treated versus control groups: T/C percent = (mean tumor volume of
53412 v2/DC \ ζ \ compound-treated group on day X / mean tumor volume of control group on day X) x 100, the optimal value, being the minimal T/C ratio which reflects the maximal tumor growth inhibition achieved and (//) Specific Tumor Growth Delay (SGD), calculated as [Td (compound-treated group)-Td (vehicle-treated group)] / Td (vehicle-treated group), with Td being the tumor doubling time of compound-treated and control groups, defined as the time in days required for the tumor volume to double.
[00339] Various HSP90 inhibitors are tested in cisNfl ;p53 mouse models developed previously (Vogel et al, 1999, Science 286: 2176-2179; Cichowski et al., 1999 Science. 286: 2172-2176). MPNSTs can harbor mutations in at least 2 tumor suppressor genes in addition to the NFl loss of heterozygosity. In particular, several studies have reported mutations in the p53 gene. The Nfl;p53 mice develop tumors with a 100% incidence between 5 and 6 months of age. In all cases the tumors exhibit characteristics of NFl associated malignancies of the peripheral nervous system, including MPNSTs, triton tumors and rhabdomyosarcomas, according to histopathological criteria. The full penetrance and reproducibility of tumor timing of appearance make the cisNfl ;p53 mouse ideal for testing therapeutic compounds since reduction in tumor burden or delay in tumor appearance can be readily monitored. [00340] Mice are randomized among control and treated groups with 15-20 mice in each experimental group. Mice are dosed every week day with high, medium, and low doses and vehicle control for 8 weeks or less beginning at 18-20 weeks of age, when 30-50% of mice begin to develop tumors. The high dose is defined as the MTD dose of the test compound with similar schedule. If toxicity is an issue for the proposed duration, lower starting dose, less frequent dosing schedule such as every other day, twice a week, or increase the sample size per group are implemented so that a sufficient number of live mice are evaluated at the end of the study. Test agents are administered via intraperitoneal (i.p.), intravenous (i.v.), or oral route. In case of significant variations in body weight during the treatment period, doses are adjusted accordingly or experiments can be repeated with the adjusted doses. [00341] Typically, cisNfl ;p53 mice that develop tumors become less active and assume classic rounded positions and ruffled coat. An untreated cohort of mice is expected to begin to develop signs of ill health within the fifth month of age. Pairs of control and test mice are sacrificed on a weekly basis once the signs of illness become apparent in control mice. All mice are carefully necropsied and the number, location and size of all tumors are noted. All tumor samples are evaluated for histologic and immunohistochemical analysis to determine pathology as well as evidence for apoptosis, necrosis, and proliferation.
53412 v2, DC ] 52 [00342] For compound efficacy analysis, the mean tumor volume of each dosing group is compared to that of the vehicle control group. Student's t-test is used for statistical analysis of tumor volume. Differences with a p-value <0.05 is considered as statistically significant. [00343] It is understood that the present invention is not limited to the particular methods and components, etc, described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein, the singular forms "a," "an,'" and "the" include the plural reference unless the context clearly dictates otherwise.
[00344] Experimental procedures and characterization data for exemplary compounds follows. The compounds and data described below are understood to be non-limiting.
Figure imgf000154_0001
2-95a 2-95c 2-95J
[00345] General procedure for oxidation using solvent system A: A solution of aldehyde 2- 94 (1.0 equiv.) in H2O/THF/DMSO (20: 10: 1 , 0.03 M) was sequentially treated at 0 0C with sulfamic acid (3.5 equiv.) and a solution of sodium chlorite (3.25 equiv.) in H2O. After 0.5-1 h stirring at this temperature, the reaction mixture was diluted with Et2O, washed with saturated NH4Cl^ and dried over MgSO4. Concentration under reduced pressure afforded the corresponding acid 2-95 which was used without any further purification in the next step.
2-95c: 1H NMR (400 MHz, (CD3)2CO, 25 0C): 5 = 6.48 (d, J = 2.2 Hz, I H), 6.45 (d, J = 2.2 Hz, I H), 3.84 (s, 3H), 3.83 (s, 3H), 2.33 (s, 3H).
2-94J: 1H NMR (400 MHz, (CDj)2CO, 25 0C): 6 = 7.58 (d, J = 7.0 Hz, 4H), 7.47-7.35 (m, 10H), 7.31-7.21 (m, 6H), 6.31 (s, I H), 5.89 (s, I H), 2.35 (s, 3H), 1.05 (s, 9H), 0.93 (s, 9H).
Figure imgf000154_0002
53412 v2 DC 153 [00346] General procedure for oxidation using solvent system B: A solution of aldehyde 2-94 (1.0 equiv.) in H2O/THF (20: 10, 0.03 M) was sequentially treated at 0 0C with sulfamic acid (3.5 equiv.) and a solution of sodium chlorite (3.25 equiv.) in H2O. After 12 h stirring at room temperature, the reaction mixture was diluted with Et2O, washed with saturated NH4Claq. and dried over MgSO4. Concentration under reduced pressure afforded the corresponding acid 2- 95 which was used without any further purification in the next step. For 2-95b, 2.0 equiv. of sulfamic acid were necessary to avoid over-chlorination. For 2-95i and 2-95k, the reaction was complete after 30 min at 0 0C.
2-95b: 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ = 6.48 (s, I H), 2.70 (s, 3H).
2-95d: 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ - 6.77 (s, I H), 3.97 (s, 3H), 3.89 (s, 3H), 2.35 (s, 3H).
2-95h: 1H NMR (400 MHz, (CD3J2CO, 25 0C): δ = 6.71 (s, IH), 5.43 (s, 2H), 3.85 (t, J = 8.2 Hz, 2H), 2.71 (s, 3H), 1.00 (t, J = 8.2 Hz, 2H), 0.04 (s, 9H).
2-951. 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ = 7.82-7.80 (m, 4H), 7.55-7.48 (m, 6H), 6.02 (s, 1 H), 2.74 (s, 3H), 1.17 (s, 9H).
2-95k: 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ = 7.48-7.34 (m, 12H), 7.23-7.16 (m, 8H), 5.93 (s, IH), 2.54 (s, 3H), 1 .05 (s, 9H), 1.02 (s, 9H).
Figure imgf000155_0001
2-95e 2-95g
[00347] General procedure for oxidation using solvent system C: To a solution of aldehyde 2-94 (1.0 equiv.) in DMSO (0.4 M) at 0 0C, were added slowly in a sequential fashion,
NaH2PO4^H2O (5.0 equiv.) dissolved in H2O (3 M) and NaClO2 (5.0 equiv.) dissolved in H2O (3 M). After stirring for 12 h, the reaction was diluted with Et2O, washed with saturated NH4Claq. and dried over MgSO4. Concentration under reduced pressure resulted into the corresponding acid 2-95 used without further purification in the next step. 2-95e; 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ = 6.76 (d, J = 1.9 Hz, I H), 6.60 (d, J = 1.6 Hz, IH), 5.25 (s, 4H), 3.75-3.69 (m, 4H), 2.31 (s, 3H), 1.18 (t, J= 7.0 Hz, 6H).
53412 v2/DC ] 54 2-95g: 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ = 12.13 (bs, IH), 6.46 (s, 2H), 5.30 (s, 2H), 3.73 (q, J= 7.0 Hz, 4H), 2.58 (s, 3H), 1.19 (t, J= 7.0 Hz, 3H).
Figure imgf000156_0001
1 -Chloro-2,4-bis-ethoxymethoxy-7-methyl-7,8, 1 1 , 12-tetrahydro- 16H-6-oxa- benzocyclotetradecene-5,15-dione (2-1 12): A 2 mM solution of compound 2-111 (200 mg, 0.38 ramol) in anhydrous toluene (190 mL) was treated with 10 % mol of catalyst Grubbs' II (30 mg, 0.038 mmol) and heated at 80 0C overnight. The reaction mixture was then passed through a pad of silica, which was washed with CH2Cl2. The combined filtrates were concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-25 % EtOAc/hexane gradient) afforded pure macrocycle 2-112 (167 mg, 94 %). 1H NMR (400 MHz, C6D6, 25 0C): δ = 7.27 (s, 1 H), 6.85 (dt, J = 15.2, 7.6 Hz, 1 H), 6.15 (d, J = 15.8 Hz, I H), 5.16-4.94 (m, 7H), 4.41 (d, J = 17.0 Hz, I H), 4.13 (d, J = 17.0 Hz, I H), 3.61-3.45 (m, 4H), 2.18-2.07 (m, 2H), 1.86- 1.62 (m, 4H), 1.38 (d, J = 5.8 Hz, 3H), 1.1 1 (t, J = 7.0 Hz, 3H), 1.04 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 193.5, 166.4, 155.0, 154.1, 146.0, 133.7, 131.5, 128.8, 127.7, 121.2, 1 18.1 , 102.9, 93.7, 93.6, 71.6, 64.4, 64.4, 45.0, 39.2, 30.7, 30.4, 19.3, 14.9, 14.8; LR. (film): vmax = 2917, 1720, 1690, 1622, 1591 , 1320, 1255, 1 120, 1037 cm"1 ; HRMS (ESI-TOF): m/z: calculated for C24H31O7ClNa: 489.1651 , found 489.1737 [Λf+Naτ]. (-)-(2R): [α]25 D = - 24.0 (c 0.59, CHCl3).
Figure imgf000156_0002
2-85 Pochonin D
Pochonin D (2-85): Compound 2-112 (50 mg, 0.1 mmol) was stirred for 2 h in a 5: 1 mixture of CH2CI2/TFA (3 mL). Concentration under reduced pressure, followed by flash chromatography (silica gel, 0-33 % EtOAc/hexane gradient) afforded synthetic pochonin D 2- 85 (25 mg, 72 %). Synthetic pochonin D was found to have identical 1H NMR as natural pochonin D. 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.42 (s, I H), 6.89 (s, I H), 6.67-6.62 (m, IH), 5.82 (d, J= 15.6 Hz, IH), 5.17-5.12 (m, IH), 5.00-4.92 (m, IH), 4.76-4.69 (m, IH), 4.28
53412 v2/DC J 55 (d, J = 17.2 Hz, IH), 4.18 (d, J = 17.7 Hz, IH), 2.54-2.47 (m, IH), 1.93-1.77 (m, 5H), 0.98 (d, J = 6.4 Hz, 3H); 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.73 (s, IH), 6.76-6.69 (m + s, 2H), 6.19 (s, IH), 5.82 (d, J = 15.2 Hz, IH), 5.46-5.40 (m, IH), 5.31-5.15 (m, 2H), 4.37 (d, J = 17.6 Hz, I H), 4.09 (d, J = 17.0 Hz, I H), 2.68-2.61 (m, I H), 2.39-2.03 (m, 5H), 1.34 (d, J = 7.0 Hz, 3H); 1H NMR (400 MHz, CD3OD, 25 0C): δ = 6.74 (dt, J = 15.5, 7.6 Hz, IH), 6.51 (s, I H), 5.83 (d, J = 15.5 Hz, I H), 5.36-5.22 (m, 3H), 4.25 (d, J = 17.7 Hz, I H), 4.13 (d, J = 17.7 Hz, 1 H), 2.54-2.47 (ddd, J = 14.5, 8.0, 4.0 Hz, 1 H), 2.31 -2.15 (m, 5H), 1.31 (d, J = 6.6 Hz, 3H);13C NMR ( 100 MHz, C6D6, 25 0C): δ = 194.2, 169.9, 164.3, 157.3, 146.1, 137.1 , 131.9, 128.1 , 126.2, 1 15.5, 107.5, 103.6, 72.4, 45.0, 36.4, 31.0, 30.8, 17.2; LR. (KBr): vmax = 2936, 1654, 1603, 1347, 1313, 1239 cm"1 ; HRMS (ESI-TOF): m/z: calculated for C18H19O5ClNa: 373.0813, found 373.0903 [M+Na*]. (+)-(2R): [α]25 D = +1 1.1 (c 0.72, CHCl3),
Figure imgf000157_0001
Hepta-2,6-dienoic acid methoxy-methyl-amide (2-1 14): To a solution of 2-chloro-yV-methoxy- vV-methylacetamide (6.0 g, 48.8 mmol) in dry DMF (20 mL) at 23 0C was added 3- mercaptophenol (4.44 mL, 48.8 mmol) and K2CO3 (6.7 g, 48.8 mmol). The resulting suspension was stirred at 23 0C overnight. After this period of time, Merrifield resin (24 g, < 2 mmol.g"1, < 48.8 mmol) was added to the mixture followed by K2CO3 (1 1.4 g, 83.0 mmol) as well as TBAI (catalytic amount), and the suspension was heated up to 50 0C. After 12 hours at this temperature, the resin was filtered and washed several times: HClaq, (50 mL), MeOH (50 mL), CH2Cl2 (50 mL) and Et2O (50 mL). The resin was dried under reduced pressure to constant mass of 29.2 g. The final mass gain (5.2 g, 27.3 mmol) indicated an estimate loading of 0.81 mmol.g"1. Resin 2-49 (10 g, 0.81 mmol.g"1) was suspended in a 1 : 1 mixture Of HFIPZCH2Cl2 (50 mL). To this suspension, H2O2 (3 mL, 16.0 mmol) was added at 23 0C and the resulting mixture was shaken for 12 h. Resin 2-113 was then filtered, washed using MeOH (50 mL), CH2Cl2 (50 mL) and Et2O (50 mL) and dried under reduced pressure to constant mass before subsequent use. Resin 2-113 (4.0 g, < 0.81 mmol.g" 1) was suspended in DMSO (40 mL) followed by the addition of fBuOK (336 mg, 3.0 mmol). After shaking the reaction for 1 h at room temperature, 5-iodo-l-pentene (588 mg, 3.0 mmol) was added to the suspension and the mixture was shaken for 3 h. The resin was filtered, washed and dried as before. Then, it was suspended in toluene and heated at 80 0C. After 8 h at this temperature,
53412 V2-DC 155 the resin was filtered and washed several times with more toluene. The combined toluene solutions were evaporated giving pure compound 2-114 as a colourless oil (321 mg, 77 %) of 95 % purity judged by NMR. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.94 (dt, J= 15.7, 6.7 Hz, IH), 6.39 (d, J = 15.2 Hz, IH), 5.84-5,74 (m, IH), 5.02 (dd, J = 17.4, 1.7 Hz, IH), 4.97 (d, J = 10.1 Hz, IH), 3.67 (s, 3H), 3.21 (s, 3H), 2.34-2.29 (m, 2H), 2.23-2.18 (m, 2H); 13C NMR (I OO MHz. CDCl3, 25 0C): δ = 166.8, 146.7, 137.3, 1 19.1, 1 15.3, 61.6, 32.3, 31.7, (one carbon is not detected); LR. (film): vmaλ = 2934, 1681 , 1638, 1378, 1 179 cm'1.
Figure imgf000158_0001
2-120
2,4-Bis-ethoxymethoxy-7-methyl-7,8, 11 ,12-tetrahydro- 1 όH-6-oxa-benzocyclotetradecene- 5,15-dione (2-120). A 2 mM solution of crude 2-119 (1.5 mmol) in anhydrous toluene (750 mL) was treated with 10 % mol of catalyst Grubbs' II (139 mg, 0.15 mmol), and heated at 80 0C overnight. The crude reaction mixture was then passed through a pad of silica, which was washed with CH2CI2. The combined filtrates were concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-25 % EtOAc/cyclohexane gradient) afforded pure 2-120 (260 mg, 40 % over two steps). 1H NMR (400 MHz. C6D6, 25 0C): δ = 7.08 (d, J = 2.2 Hz, I H), 7.01 (d, J = 2.2 Hz, I H), 6.93-6.86 (m, I H), 6.17 (d, J = 16.1 Hz, I H), 5.34-4.90 (m, 7H), 4.41 (d, J = 14.5 Hz, IH), 3.75 (d, J = 14.5 Hz, IH), 3.59-3.45 (m, 4H), 2.27-2.12 (m, 2H), 1.95-1.61 (m, 4H), 1.45 (d, J = 6.2 Hz, 3H), 1.07 (t, J = 7.0 Hz, 3H), 1.07 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 196.0, 167.2, 159.5, 156.6, 147.6, 135.8, 131.5, 130.2, 128.5, 1 19.1 , 109.7, 102.3, 93.3, 92.9, 71.0, 64.2, 64.0, 44.5, 39.6, 30.9, 30.2. 20.1 , 14.8, 14.8; LR. (film): vmax = 2976, 1717, 1602, 1438, 1284, 1 155, 1 1 10, 1036, 1018 cm" 1; HRMS (ESI-TOF): m/∑: calculated for C24H32O7Na: 455.2040, found 455.2135 [Λ/+Na~]. (-)-(2Λ): [α]25 D = - 50.3 (c 1.00, CHCl3).
Figure imgf000158_0002
2-85 Pochonin D
53412 v2 'DC 1 57 Pochonin D (2-85) using polymer-bound reagents: PS-TsOH (300 mg, 3.2 mmol.g"1) was added to a solution of compound 2-112 (50 mg, 0.1 mmol) in MeOH (3 mL) of and the suspension was shaken at 40 0C for 4 h. The reaction mixture was then filtered and the methanolic solution concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-20 % EtOAc/cyclohexane gradient) afforded synthetic pochonin D 2-85 (32 mg, 90 %).
Figure imgf000159_0001
2-103 Monocillin Il
Monocillin II (2-103) using polymer-bound reagents: PS-TsOH (145 mg, 3.2 mmol.g"1) was added to a solution of compound 2-120 (20 mg, 0.05 mmol) in MeOH (1.5 mL) and the suspension was shaken at 40 0C for 4 h. The reaction mixture was then filtered and the methanolic solution concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-20 % EtOAc/cyclohexane gradient) afforded synthetic monocillin II 2-103 (14 mg, 92 %). 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.49 (s, I H), 6.70-6.62 (m, I H), 6.49 (d, J = 2.9 Hz, IH), 6.09 (d, J = 2.4 Hz, IH), 5.86 (d, J = 15.2 Hz,
IH), 5.08-4.95 (m, 2H), 4.82-4.75 (m, IH), 4.14 (d, J = 16.8 Hz, IH), 3.71 (d, J = 17.0 Hz,
I H), 2.64-2.57 (m, IH), 1.83-1.76 (m, 3H), 1.74-1.66 (m, 2H), 0.97 (d, J = 6.4 Hz, 3H); 13C
NMR (100 MHz, C6D6, 25 0C): δ = 195.7, 170.3, 166.5, 161.3, 146.1, 140.5, 131.7, 129.9,
126.5, 1 12.4, 102.8, 72.2, 49.1 , 36.6, 31.0, 30.6, 17.5, (one carbon is not detected); LR. (KBr): vniax = 2936, 1654, 1603, 1347, 1313, 1239 cm" 1; HRMS (ESI-TOF): m/z: calculated for Ci8H2IO5 requires 317.3980, found 317.3978 [M+H+]. (+)-(2Λ): [α]25 D = + 40.6 (c 0.18,
CHCl3).
Figure imgf000159_0002
2-123
Macrocycle 2-122: To a solution of compound 2-112 (50 mg, 0.1 1 mmol) in CH3CN (5 mL) at 0 0C was added freshly made DMDO (275 μL, 0.1 1 mmol, 0.04 M in acetone) and the
53412 v2/DC 15g mixture was stirred for 1.5 h. After evaporation of the solvents under reduced pressure, purification by flash chromatography (silica gel, 0-70 % Et2θ/hexane gradient) afforded compound 2-122 (41 mg, 79 %) as a 1 :1 mixture of two diastereoisomers. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.12 (s, IH), 7.1 1 (s, IH), 6.90-6.76 (m, 2H), 6.11 (d, J = 15.6 Hz, IH), 6.05 (d, J = 15.8 Hz, IH), 5.37-5.27 (m, 6H), 5.26-5.21 (m, 4H), 4.14 (d, J = 16.9 Hz, IH), 4.12 (d, J = 17.4 Hz, IH), 4.04 (d, J = 17.2 Hz, IH), 3.82-3.70 (m, 9H), 2.81-2.78 (m, I H), 2.74-2.72 (m, I H), 2.67-2.62 (m, 2H), 2.38-2.1 1 (m, 8H), 2.05-2.03 (m, IH), 2.03-2.00 (m, I H), 1.74-1.60 (m, 2H), 1.41 (d, J = 7.2 Hz, 3H), 1 ,39 (d, J = 6.2 Hz, 3H), 1.27-1.22 (m, 12H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 195.1 (x 2), 166.7, 166.3, 154.9, 154.8, 154.0, 153.5, 147.8, 147.4, 132.9, 132.4, 129.1 , 129.0, 1 19.7 (x 2), 1 18.0, 1 17.9, 102.9, 102.8, 93.9 (x 2), 93.6 (x 2), 71.1 , 70.4, 64.8 (x 2), 64.6 (x 2), 58.4, 57.6, 56.9, 55.1 , 43.4 (x 2), 39.0, 37.9, 29.9, 29.7, 27.9 (x 2), 18.4, 18.0, 15.0 (x 4); HRMS (ESI-TOF): m/z: calculated for C24H32ClO8: 483.1780, found 483.1814 [M+W].
Figure imgf000160_0001
[00348] Macrocycles 2-124 and 2-125: PS-TsOH (264 mg, 3.2 mmol.g" 1) was added to a solution of compound 2-123 (41 mg, 85 μmol) in MeOH (3 mL) and the suspension was shaken at 40 0C until consumption of all starting material (~1 h). The reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. L.C./M.S. analysis of the crude mixture showed clearly 2 peaks corresponding to methanol addition on the conjugated olefin (2-124) and opening of the epoxide as a diol (2-125).
2-125: 1H NMR (400 MHz, CD3OD, 25 0C): δ = 7.19 (m, IH), 6.89-6.81 (m, IH), 6.52 (s, I H), 6.47 (s, I H), 6.20 (d, J = 16.1 Hz, I H), 6.04 (d, J = 15.6 Hz, IH), 5.54-5.49 (m, IH), 5.43-5.36 (m, I H), 4.50 (d, J = 17.7 Hz, IH), 4.46 (d, J= 17.7 Hz, IH), 4.39 (d, J = 17.2 Hz, IH), 4.07 (d, J = 17.2 Hz, IH), 3.80-3.64 (m, 2H), 3.51-3.46 (m, 2H), 2.62-2.58 (m, IH), 2.39-2.30 (m, 2H), 2.27-2.18 (m, I H), 2.08-2.00 (m, 2H), 2.00-1.85 (m, 4H), 1.44 (d, J = 6.4 Hz, 6H); HRMS (ESI-TOF): m/z: calculated for C]8H22ClO7: 385.1054, found 385.0944 [M+¥T],
2-124: compound 2-124 which was characterized as the MeOH-addition on the α,β- conjugated system based on the loose of olefinic protons in the NMR (a detailed assignment
53412 V2/DC 159 is not possible as product 2-124 represents a mixture of 4 compounds); HRMS (ESI-TOF): m/z: calculated for C19H24ClO7: 399.121 1 , found 399.1030 [M+H+],
Figure imgf000161_0001
2-128 Macrocycle 2-128: A 2 mM solution of compound 2-127 (140 mg, 0.23 mmol) in anhydrous toluene (1 15 mL) was treated with 10 % mol of catalyst Grubbs' II (18.4 mg, 0.023 mmol) and heated up to 80 0C for 12 h. The reaction mixture was then filtered through a pad of silica, which was washed with CH2Cl?. The combined filtrates were concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-25 % EtOAc/hexane gradient) afforded macrocycle 2-128 (1 16 mg, 87 %). 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.71 (dt, J = 15.3, 7.3 Hz, IH), 6.45 (s, IH), 5.81 (d, J = 15.3 Hz, IH), 5.25 (s, 2H), 5.04- 5.03 (m, I H), 3.89 (d, J = 17.4 Hz, I H), 3.57 (d, J= 17.4 Hz, IH), 2.31-2.04 (m, 6H), 1.35 (d, J = 6.4 Hz, 3H), 1.03 (s, 9H), 0.99 (s, 9H), 0.28-0.24 (m, 12H); i 3C NMR (100 MHz, CDCl3, 25 0C): 6 = 195.8, 166.8, 152.9, 151.7, 146.5, 132.7, 131.9, 128.6, 126.8, 122.8, 119.7, 110.7, 71.9, 45.6, 38.5, 30.9, 25.7 (x 4), 25.6 (x 4), 18.7, 18.3, -4.1 (x 2), -4.4 (x 2) ; HRMS (ESI- TOF): m/z: calculated for C30H47ClO5Si2Na: 601.2543, found 601.2568 [M+Naτ].
Figure imgf000161_0002
2-129
Macrocycle 2-129: An aqueous Na2^EDTA solution (700 μL, 4 x 10"4M) was added to a solution of compound 2-128 (80 mg, 0.14 mmol) in a 2: 1 mixture of dimethoxymethane/acetonitrile (2.1 mL). The resulting mixture was cooled to 0 0C and treated with trifluoroacetone (150 μL) added via a precooled syringe. A mixture of sodium bicarbonate (88 mg, 1.05 mmol) and Oxone (430 mg, 0.70 mmol) was added in portions over a period of ~1 h to this homogeneous solution. The reaction was followed by TLC and found to be complete in 2 h. The reaction mixture was then poured into water (10 mL), extracted with CH2Cl2 (20 mL) and dried over Na2SO4. Removal of the solvent under reduced pressure afforded pure compound 2-129 (66 mg, 93 %) as a mixture of 2 diastereoisomers in a 3: 1 ratio. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.90-6.77 (m, 1.33H), 6.45 (s, 1.33H), 6.06 (d,
53412 v2 DC 1 (30 J = 15.8 Hz, 1.33H), 5.31-5.29 (m, IH), 5.29-5.21 (m, O.33H), 4.03 (d, J = 18.1 Hz, 1.33H), 3.63 (d, J = 17.6 Hz, IH), 2.82-2.80 (m, 1.33H), 2.74-2.71 (m, 1.33H), 2.62-2.60 (m, 1.33H), 2.41-2.1 1 (m, 4.6H), 2.02-1.95 (m, 0.33H),1.80-1.78 (m; IH), 1.78-1.68 (m, 0.7H), 1.41 (d, J = 6.4 Hz, 3.9H), 1.05 (s, 12H), 0.97 (s, 12H), 0.26 (s, 16H); 13C NMR (100 MHz, CDCl3, 25 0C): 6 = 195.4, 195.1 , 167.0, 166.2, 153.1 (x 2), 152.1, 151.5, 147.8, 146.9, 132.9, 132.2, 129.0, 128.3, 122.2, 121.4, 120.0, 1 19.7, 1 10.6, 1 10.3, 71.3, 70.1 , 58.2, 57.9, 56.6, 55.2, 44.7, 43.9, 38.7, 37.6, 30.1 , 29.4, 28.3, 27.5, 25.7 (x 4), 25.6 (x 4), 25.5 (x 4), 25.4 (x 4), 20.8, 17.8, -3.9, -4.0, -4.3 (x 3), -4.4 (x 3); HRMS (ESI-TOF): m/z: calculated for C30H48O6ClSi2: 595.2672, found 595. 2698 [M+H÷].
Figure imgf000162_0001
2-122 Pochonin A
Pochonin A (2-122): TBAF (244 μL, I M solution in hexane, 0.24 mmol) was added to a solution of compound 2-129 (66 mg, 0.1 1 mmol) in THF (2 mL) and the mixture was stirred at room temperature for 20 min. The reaction was then quenched with saturated NH4Claq. (8 mL), extracted several times with EtOAc (10 mL) and dried over Na2SO4. Concentration under reduced pressure followed by purification by flash chromatography (silica gel, 0-70 % EtiO/hexane) afforded two different diastereoisomers pochonin A (2-122) and its diastereoisomer 2-122b as a 3: 1 mixture (80 % yield) The isomers were separated by preparative TLC with a 3: 1 mixture of Et2O/hexane. 1H NMR (400 MHz, [D6]DMSO, 25 0C): δ = 10.81 (s, I H), 10.74 (s, IH), 6.97-6.89 (m, I H), 6.53 (s, I H), 6.08 (d, J = 15.8 Hz, 1 H), 5.15-5.13 (m, 1 H), 4.19 (d, J = 17.5 Hz, 1 H), 4.09 (d, J = 17.5 Hz, 1 H), 2.81 (s, 1 H), 2.60 (m, I H), 2.44-2.40 (m, 2H), 2.30-2.22 (m, 2H), 1.80-1.78 (m, 2H), 1.32 (d, J = 6.4 Hz, 3H); 1H NMR (400 MHz, CDCl3, 25 0C): δ = 11.85 (s, I H), 6.94-6.87 (m, I H), 6.70 (s, IH), 6.14 (s, I H), 6.12 (d, J = 16.4 Hz, IH), 5.32-5.31 (m, IH), 4.53 (d, J= 18.1 Hz, IH), 4.27 (d, J = 18.1 Hz, I H), 2.77 (s, I H), 2.58-2.56 (m, 2H), 2.47-2.43 (m, IH), 2.35-2.28 (m, IH), 2.1 1 -2.07 (m, I H), 1.93-1 .86 (m, I H), 1.51 (d, J = 6.4 Hz, 3H), 0.94-0.90 (m, IH); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 195.0, 170.0, 164.1 , 156.4, 147.5, 135.7, 129.9, 1 15.0, 107.3, 103.8, 72.2, 57.0, 55.5, 45.1 , 36.3, 30.9, 29.1 , 17.9; HRMS (ESI-TOF): m/z: calculated for Ci8H19ClO6Na: 389.0762, found 389.0724 [M+Na+], (-)-(2R, 4R, 5R): [α]25 D = - 7.0 (c 0.1 1, CHCl3).
53412 v2/DC \ β \
Figure imgf000163_0001
Compound 2- 122b: 1H NMR (400 MHz, [D6]DMSO, 25 0C): δ = 10.74 (s, IH), 10.39 (s, I H), 6.94-6.89 (m, I H), 6.52 (s, I H), 6.02 (d, J = 16.4 Hz, IH), 5.18 (m, I H), 4.32 (d, J = 17.5 Hz, IH), 3.96 (d, J = 17.5 Hz, IH), 2.82 (s, IH), 2.68 (s, IH), 2.34-2.26 (m, 3H), 1.86- 1.83 (m, IH), 1.70-1.63 (m, I H), 1.22 (d, J= 5.8 Hz, 3H), IH masked by the solvent peak; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.37 (s, IH), 6.90-6.83 (m, IH), 6.67 (s, IH), 6.24 (d, J = 16.4 Hz, IH), 6.08 (s, IH), 5.39-5.37 (m, IH), 4.52-4.36 (m, 2H), 2.72-2.62 (m, 2H), 2.56- 2.52 (m, I H), 2.45-2.40 (m, IH), 2.40-2.37 (m, IH), 2.08-2.04 (m, IH), 1.91-1.86 (m, IH), 1.35 (d, J = 6.4 Hz, 3H), I H masked by the solvent peak; HRMS (ESI-TOF): m/z: calculated for Ci8H19ClO6Na: 389.0762; found 389.0796 [M+Na+], (+)-(2R, 4S, 55): [α]25 D = + 13.8 (c 0.13, CHCl3).
Figure imgf000163_0002
2-132
Macrocycle 2-132: A 2 niM solution of compound 2-131 (166 mg, 0.26 mmol) in anhydrous toluene (130 mL) was treated with 10 % mol of Grubbs' II (20.8 mg, 0.026 mmol) and heated up to 80 0C for 12 h. The reaction mixture was then filtered through a pad of silica, which was washed with CH2Cl2. The combined filtrates were concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-25 % EtOAc/hexane gradient) afforded macrocycle 2-132 (136 mg, 87 %). 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.08 (s, IH), 6.77-6.71 (m, I H), 5.89 (d, J = 15.2 Hz, IH), 5.35-5.24 (m, 6H), 5.09-5.05 (m, IH), 4.02 (d, J = 17.0 Hz, I H), 3.85-3.78 (m, 5H), 2.37-2.08 (m, 6H), 1.39 (d, J = 5.8 Hz, 3H), 1.02-0.97 (m, 4H), 0.04 (s, 18H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 195.7, 166.8, 154.6, 153.9, 147.0, 132.8, 131.7, 128.6, 127.5, 120.6, 1 17.8, 102.7, 93.7, 93.3, 71.9, 66.9, 66.6, 44.7, 39.2, 30.8 (x 2), 19.5, 18.0, 17.9, -1.4 (x 6); HRMS (ESI-TOF): m/z: calculated for C30H47O7ClSi2H2O: 628.2649, found 628.2870 [MfH2O]. (-)-(2R): [α]25 D = - 16.3 (c 0.85, CHCl3).
53412 V2-DC 162
Figure imgf000164_0001
Macrocycle 2-133: An aqueous Na2*EDTA solution (350 μL, 4 x 10"4M) was added to a solution of compound 2-132 (40 mg, 65 μmol) in a 2: 1 mixture of dimethoxymethane/acetonitrile (1.1 mL). The resulting solution was cooled to 0 0C and trifluoroacetone (75 μL) was added via a precooled syringe. A mixture of sodium bicarbonate (44 mg, 0.5 mmol) and Oxone (215 mg, 0.35 mmol) was then added in portions over a period of ~1 h to this homogeneous solution. The reaction was followed by TLC and found to be complete in 2 h. The reaction mixture was then poured into water (5 mL), extracted with CH2Cl2 (10 mL) and dried over Na2SC>4. Removal of the solvent under reduced pressure afforded pure compound 2-133 (34 mg, 82 %) as a 1 : 1 mixture of two diastereoisomers. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.1 1 (s, 1 H), 7.09 (s, 1 H), 6.91 -6.76 (m, 2H), 6.12 (d, J = 15.8 Hz, 1 H), 6.06 (d, J = 15.8 Hz, 1 H), 5.36-5.31 (m, 6H), 5.23-5.22 (m, 4H), 4.15 (d, J = 16.9 Hz, I H), 4.13 (d, J = 16.0 Hz, I H), 4.05 (d, J = 17.5 Hz, I H), 3.85-3.75 (m, 9H), 2.81 - 2.79 (m, I H), 2.75-2.73 (m, I H), 2.68-2.62 (m, 2H), 2.42-2.29 (m, 10H), 1.72-1.60 (m, 2H), 1.41 (d, J = 7.6 Hz, 3H), 1.39 (d, J= 6.4 Hz, 3H), 1.02-0.96 (m, 8H), 0.03 (s, 36H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 195.1 (x 2), 166.8, 166.3, 154.9 (x 2), 154.2, 153.7, 147.8, 147.4, 132.9, 132.4, 129.1 , 129.0, 1 19.8, 1 19.6, 1 17.9, 1 17.8, 102.8, 102.7, 93.7 (x 2), 93.4 (x 2), 71.1 , 70.3, 66.9, 66.8, 66.6, 66.5, 58.4, 57.6, 56.9, 55.1 , 43.5 (x 2), 38.9, 37.9, 29.9, 29.7, 27.9 (x 2), 20.7 (x 2), 18.4, 18.0, 17.9 (x 2), -1.40 (x 12); HRMS (ESI-TOF): m/∑: calculated for C30H4xO8ClSi2: 627.2571 , found 627.2551 [M+W],
Figure imgf000164_0002
2-122 Pochoniπ A
[00349] Pochonin A (2-122) from macrocycle 2-133: A solution of compound 2-133 (21 mg, 33 μmol) in CH2CI2 (2.5 mL) was treated at room temperature with MgBr2^Et2O (69 mg, 0.27 mmol). The reaction was followed by L.C./M.S. until bromohydrine started appearing (~1 h). The reaction was then diluted with EtOAc (5 mL), washed with saturated NH4Cl3Q (5 mL) and dried over MgSO4. After concentration under reduced pressure, purification by flash
53412 v2'DC 153 chromatography (silica gel, 0-70 % Et2θ/hexane gradient) afforded pochonin A (2-122) (8.6 mg, 70 %) as a 1 : 1 mixture of diastereoisomers.
[00350] General procedure for the synthesis of compounds 2-110a-g and 2-1 17a-g: A solution of acid 2-95a or 2-95b (1.0 equiv.), homoallylic alcohol (/?)-120a-g or (S)-120a-g (1.0 equiv.) and tris-(3-chlorophenyl)phosphine (2.0 equiv.) in anhydrous toluene (0.05 M) was treated at room temperature with PS-DEAD (2.5 equiv., 1.3 mmol.g"1). After stirring for 30 min, the reaction mixture was filtered on silica and washed with hexane/EtOAc (10: 1 , 100 mL) and hexane/EtOAc (3: 1 , 100 mL). The 3 : 1 mixture was concentrated under reduced pressure to yield compound 2-115a-g or 2-116a-g (60-80 %). Without further purification, compound 2-115a-g or 2-116a-g (1.0 equiv.) and TBAI (catalytic amount) were dissolved in DMF (0.15 M) and treated with diisopropylethylamine (4.0 equiv.) and chloromethyl ethyl ether (4.0 equiv.). After stirring overnight at 80 0C, the reaction mixture was diluted with EtOAc and washed several times with a saturated NH4Claq. solution. The organic phase was dried over MgSC>4 and concentrated under reduced pressure to yield compounds 2-110a-g and 2-117a-g (80-90 %).
[00351] General procedure for the synthesis of compounds 2-1 19a-g and 2-140a-g: A solution of compound 2-110a-g or 2-117a-g (1.0 equiv.) in anhydrous THF (0.2 M) cooled at -78 0C was treated with freshly prepared LDA (2.0 equiv.). Immediately after, the α,β- unsaturated Weinreb amide 2-114 was added to the cooled solution (1.0 equiv.). The resulting mixture was then stirred for 10 min at -78 0C and quenched by addition of Amberlite resin (20 equiv.). Upon wanning up to room temperature, the reaction was filtered on a pad of silica and washed with EtOAc. Concentration under reduced pressure afforded the desired compound 2-118a-g or 2-119a-g. This compound was used directly in the metathesis reaction without any further purification. When X = H, 20 % of the corresponding 1 ,4-addition compound was observed and a fraction of the mixture was purified for characterization of compounds 2-119a-g and 2-140a-g (silica gel, 0-20 % EtOAc/hexane gradient). [00352] General procedure for the metathesis reaction: A solution of crude 2-118a-g or 2- 119a-g (or mixture 2-119a-g or 2-140a-g when X = H) in anhydrous toluene (2 niM) was treated with Grubbs' II (0.10 equiv.) and heated at 80 0C for 12 h. The reaction was cooled down to room temperature and the mixture was filtered through a pad of silica gel, washed with CH2CI? followed by a mixture EtOAc/cyclohexane 1 : 1 , and concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-25 % EtOAc/cyclohexane gradient) afforded compound 2-112a-g or 2-120a-g (and 2-121a-g) (38-70 % over two steps).
53412 v2. DC | g4
Figure imgf000166_0001
(S)-2-112
Compound (5)-2-112: 1H NMR (400 MHz, CDCl3, 250C): δ = 7.10 (s, IH), 6.75-6.71 (m, IH), 5.88 (d, J = 15.8 Hz, IH), 5.32 (s, 2H), 5.27-5.20 (m, 2H), 5.25 (s, 2H), 5.08-5.04 (m, IH), 4.01 (d, J = 17.0 Hz, IH), 3.82-3.73 (m, 5H), 2.36-2.32 (m, 2H), 2.26-2.20 (m, 3H), 2.12-2.05 (m, 2H), 1.38 (d, J= 5.8 Hz, 3H), 1.25 (t, J= 7.0 Hz, 3H), 1.24 (t, J= 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 250C): δ = 195.5, 166.7, 154.6, 153.8, 147.1, 132.8, 131.6, 128.6, 127.5, 120.7, 117.8, 103.0,94.0,93.7,72.0,64.8,64.6,44.7,39.1,30.8, 19.4, 15.0, 2C missing; HRMS (ESI-TOF): m/∑: calculated for C24H3IU7ClNa: 489.1551, found 489.1651 [M+Na+]. (+)-(2S): [α]25 D = + 25.0 (c 1.00, CHCl3).
Figure imgf000166_0002
Compound (R)-2-\ 12a: 1H NMR (400 MHz, CDCl3, 250C): δ = 7.49-7.47 (m, 2H), 7.40-7.29 (m, 3H), 7.10(s, IH), 6.84-6.77 (m, IH), 5.98 (d, J= 15.2Hz, IH), 5.78 (d, J= 8.8 Hz, IH), 5.44-5.30 (m, 4H), 5.15 (d, J = 7.0 Hz, 1 H), 5.05 (d, J = 6.8 Hz, 1 H), 4.07 (d, J = 17.0 Hz, IH), 3.90 (d, J= 17.0 Hz, IH), 3.80 (d, J= 7.0 Hz, 2H), 3.60-3.51 (m, 2H), 2.68-2.62 (m, IH), 2.50-2.47 (m, IH), 2.38-2.29 (m, 2H), 2.14-2.02 (m, 2H), 1.25 (t, J= 7.0 Hz, 3H), 1.17 (t, J= 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 250C): δ = 195.7, 166.7, 154.8, 154.2, 147.3, 140.7, 133.3, 132.1, 128.5, 128.3 (x 2), 128.2, 127.9, 127.7, 126.7 (x 2), 120.1, 118.1, 102.9, 93.9, 93.4, 77.4, 64.8, 64.4, 44.5, 40.5, 30.7, 15.0, 14.9; HRMS (ESI-TOF): m/r. calculated for C29H33O7ClNa: 551.1680, found 551.1807 [M+Na+]. [α]25 D = - 40.4 (c 0.79, CHCl3).
53412 v2/DC 165
Figure imgf000167_0001
(S)-2-112a
Compound (5)-2-l 12a: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.53-7.51 (m, 2H), 7.44-7.33 (m, 3H), 7.14 (s, I H), 6.88-6.81 (m, IH), 6.02 (d, J = 15.2 Hz, IH), 5.78 (dd, J = 10.5, 1.8 Hz, I H), 5.46-5.33 (m, 4H), 5.19 (d, J = 7.0 Hz, IH), 5.09 (d, J = 7.6 Hz, IH), 4.10 (d, J = 17.0 Hz, I H), 3.94 (d, J = 17.5 Hz, IH), 3.83 (d, J = 7.0 Hz, 2H), 3.64-3.55 (m, 2H), 2.70- 2.64 (m, I H), 2.54-2.50 (m, IH), 2.37-2.33 (m, 2H), 2.15-2.08 (m, 2H), 1.29 (t, J = 7.3 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 195.7, 166.7, 154.8, 154.2, 147.3, 140.7, 133.2, 132.1 , 128.5, 128.2, 127.9, 127.7, 126.7, 120.1, 1 18.0, 102.8, 93.9, 93.5, 77.4, 64.8, 64.4, 44.5, 40.5, 30.7, 15.0, 14.9, (one carbon is not detected); HRMS (ESI-TOF): m/∑; calculated for C29H33O7ClNa: 551.1680, found 551.1704 [M+Na+]. [α]25 D = + 48.8 (c 1.00, CHCl3).
Figure imgf000167_0002
Compound (R)-IA 12d: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.14 (s, IH), 6.72-6.66 (m, I H), 5.88 (d, J = 15.2 Hz, I H), 5.33-5.17 (m, 6H), 4.92-4.88 (m, IH), 4.21 (d, J = 17.0 Hz, IH), 3.92 (d, J = 17.0 Hz, IH), 3.79-3.67 (m, 4H), 2.33-2.17 (m, 5H), 2.07-1.96 (m, 2H), 1.23 (t, J = 7.0 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H), 1.00 (d, J = 5.8 Hz, 6H); 13C NMR (100 MHz, CDCl,, 25 0C): δ = 195.7, 167.1 , 154.7, 154.4, 147.4, 133.7, 131.2, 128.8, 128.4, 1 19.7, 1 18.0, 102.7, 93.9, 93.5, 80.0, 64.8, 64.5, 44.1 , 32.3, 31.2, 30.7, 30.6, 18.3, 17.2, 15.0, 14.9; HRMS (ESI-TOF): m/z: calculated for C26H35O7ClNa: 517.1964, found 517.1844 [M+Naτ]. [α]2'D = + 21.3 (c 1.00, CHCl3).
Figure imgf000167_0003
2-112g
534 l2 v2/DC ] ββ Compound 2-1 12g: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.12 (s, IH), 6.76-6.70 (m, I H), 5.87 (d, J = 15.0 Hz, I H), 5.33 (s, 2H), 5.26 (s, 2H), 5.24-5.16 (m, 2H), 4.25 (t, J = 5.1 Hz, 2H), 3.82-3.73 (m, 6H), 2.40-2.36 (m, 2H), 2.16-2.13 (m, 4H), 1.27-1.23 (m, 6H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 195.2, 167.2, 154.6, 153.4, 146.8, 132.3, 131.3, 128.9, 128.5, 121.0, 1 17.8, 103.0, 94.0, 93.7, 64.8, 64.7, 64.6, 45.4, 31.9, 31.0, 30.7, 15.0, 15.0; HRMS (ESI-TOF); m/z: calculated for C23H30O7Cl: 453.1675, found 453.1672 [M+W].
Figure imgf000168_0001
Compound (S)-2-120: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.81 -6.72 (m, I H), 6.74 (d, J = 1.8 Hz, I H), 6.56 (d, J = 1.8 Hz, I H), 5.98 (d, J = 15.8 Hz, IH), 5.38-5.33 (m, 2H), 5.22- 5.13 (m, 5H), 4.06 (d, J = 14.6 Hz, I H), 3.75-3.65 (m, 4H), 3.46 (d, J = 14.6 Hz, IH), 2.37- 2.22 (m, 4H), 2.18-2.02 (m, 2H), 1.39 (d, J = 5.8 Hz, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.18 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 197.4, 167.7, 159.0, 156.1, 148.9, 135.0, 131.7, 129.8, 128.5, 1 18.5, 109.7, 102.2, 93.4, 93.0, 71.5, 64.5, 64.4, 44.3, 39.5, 30.9, 30.6, 20.2, 15.0 (x 2); HRMS (ESI-TOF): m/z: calculated for C24H32O7Na: 455.2040, found 455.1901 [Λ/+Naτ]. (+)-(2S): [α]25 D = + 59.5 (c 1.00, CHCl3).
Figure imgf000168_0002
Compound (Λ)-2-120a: H NMR (400 MHz, CDCl3, 25 0C): δ = 7.56-7.54 (m, 2H), 7.41- 7.29 (m, 3H), 6.89-6.82 (m, I H), 6.78 (d, J= 2.3 Hz, IH), 6.61 (d, J= 1.8 Hz, I H), 6.06 (d, J = 16.4 Hz, I H), 5.98 (dd, J = 11.7, 2.4 Hz, IH), 5.53-5.51 (m, 2H), 5.20 (d, J = 7.0 Hz, IH), 5.17 (d, J = 6.4 Hz, I H), 5.07 (d, J = 7.0 Hz, IH), 4.96 (d, J = 7.0 Hz, I H), 4.20 (d, J = 14.6 Hz, I H), 3.73-3.68 (m, 2H), 3.54-3.45 (m, 3H), 2.71-2.66 (m, IH), 2.55-2.51 (m, IH), 2.38- 2.32 (m, 2H), 2.23-2.06 (m, 2H), 1.22 (t, J = 7.0 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 197.6, 167.4, 159.3, 156.6, 149.0, 140.8, 135.6, 132.2, 129.9, 128.5, 128.2 (x 2), 127.9, 126.9 (x 2), 117.9, 109.9, 102.3, 93.2, 93.0, 76.6, 64.4, 64.3, 44.4,
53412 v2/DC 167 40.5, 31 ,0, 30.6, 15.0, 14.9; HRMS (ESI-TOF): m/z; calculated for C29H34O7Na: 517.2197, found 517.2062 [M+N&~]. [α]25 D = - 108.3 (c 1.00, CHCl3).
Figure imgf000169_0001
Compound (S)-2-120a: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.56-7.54 (m, 2H), 7.41-7.29 5 (m, 3H), 6.90-6.82 (m, I H), 6.78 (d, J = 2.4 Hz, IH), 6.61 (d, J = 1.8 Hz, IH), 6.07 (d, J = 16.4 Hz, I H), 5.98 (dd, J = 1 1.4, 2.0 Hz, IH), 5.53-5.51 (m, 2H), 5.20 (d, J = 7.0 Hz, IH), 5.18 (d, J = 7.0 Hz, I H), 5.07 (d, J = 7.0 Hz, IH), 4.97 (d, J = 7.0 Hz, IH), 4.20 (d, J= 14.6 Hz, I H), 3.74-3.69 (m, 2H), 3.55-3.46 (m, 3H), 2.71-2.66 (m, IH), 2.55-2.52 (m, IH), 2.38- 2.33 (m, 2H), 2.23-2.09 (m, 2H), 1.22 (t, J = 7.0 Hz, 3H), 1.15 (t, J = 7.0 Hz, 3H); 13C NMR I O (100 MHz, CDCl3, 25 0C): δ = 197.6, 167.5, 159.3, 156.6, 149.0, 140.8, 135.6, 132.2, 129.9, 128.5, 128.2 (x 2), 127.9, 126.9 (x 2), 1 17.9, 1 10.0, 102.3, 93.2, 93.0, 76.6, 64.4, 64.3, 44.4, 40.5, 31.0, 30.6, 15.0, 14.9; HRMS (ESI-TOF): m/z: calculated for C29H34O7Na: 517.2197, found 517.2049 [M+Na+]. [α]25 D = + 81.6 (c 1.00, CHCl3).
Figure imgf000169_0002
15 Compound (i?)-2- 120d: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.83 (d, J = 1.7 Hz, IH), 6.79-6.72 (m, I H), 6.61 (d, J = 1.2 Hz, I H), 6.00 (d, J = 16.4 Hz, IH), 5.38-5.36 (m, 2H), 5.26-5.09 (m, 5H), 4.30 (d, J = 14.6 Hz, I H), 3.74-3.67 (m, 4H), 3.46 (d, J = 14.6 Hz, I H), 2.33-2.26 (m, 4H), 2.18-2.14 (m, 2H), 2.06-2.01 (m, I H), 1.23 (t, J = 7.0 Hz, 3H), 1.21 (t, J = 7.0 Hz, 3H), 1 .06 (d, J = 6.6 Hz, 3H), 1.05 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 0 25 0C): δ = 197.9, 167.6, 159.2, 156.9, 149.2, 136.4, 131 .5, 129.9, 129.2, 1 17.4, 109.8, 102.0, 93.3. 93.0, 78.9, 64.4 (x 2), 44.2, 33.0, 32.0, 31 .0, 30.4, 18.3, 17.2, 15.0 (x 2); HRMS (ESI- TOF): m/z: calculated for C26H36O7Na: 483.2353, found 483.2215 [M+Na~]. [α]2S D = + 52.8 (c 1 .00, CHCl3).
53412 v2/DC 168
Figure imgf000170_0001
Compound (/?)-2-120e: 1H NMR (400 MHz, CDCl3, 250C): δ = 6.77 (d, J = 1.8 Hz, IH), 6.77-6.70 (m, IH), 6.57 (d, J= 1.7 Hz, IH), 5.97 (d, J= 16.4 Hz, IH), 5.37-5.32 (m, 2H), 5.21-5.14 (m, 5H), 4.17 (d, J = 14.6 Hz, IH), 3.73-3.65 (m, 4H), 3.45 (d, J= 14.6 Hz, IH), 2.39-2.20 (m, 4H), 2.17-2.00 (m, 2H), 1.78-1.72 (m, IH), 1.69-1.60 (m, IH), 1.54-1.44 (m, 2H), 1.22 (t, J= 7.0 Hz, 3H), 1.18 (t, J = 7.0 Hz, 3H), 0.97 (d, J = 7.3 Hz, 3H); 13CNMR (100 MHz, CDCl3, 250C): δ = 197.7, 167.6, 159.1, 156.6, 149.0, 135.8, 131.5, 129.9, 128.7, 117.9, 109.8, 102.1, 93.3, 93.0, 74.5, 64.4, 64.3, 44.2, 37.3, 37.0, 31.0, 30.5, 18.2, 15.0, 14.9, 14.2. [α]25 D = - 1.3 (c 1.00, CHCl3).
Compound (Λ)-2-120f: H NMR (400 MHz, CDCl3, 250C): δ = 7.39-7.33 (m, 4H), 7.31-7.27
(m, IH), 6.82 (s, IH), 6.82-6.75 (m, IH), 6.63 (s, IH), 6.02 (d, J= 16.4 Hz, IH), 5.35-5.29 (m, 2H), 5.27-5.20 (m, 5H), 4.16 (d, J= 14.6 Hz, IH), 3.79-3.70 (m, 4H), 3.52 (d, J = 14.6 Hz, IH), 3.37 (dd, J= 13.4,4.1 Hz, IH), 2.78 (dd, J= 13.5, 9.4Hz, IH), 2.37-2.12 (m, 5H), 2.06-2.02 (m. IH), 1.26 (t, J = 7.0 Hz, 3H), 1.24 (t, J= 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, 250C): δ = 197.6, 167.8, 159.2, 156.5, 149.0, 137.3, 135.5, 131.8, 129.9, 129.5 (x 2), 128.6 (x 2), 128.4, 126.7, 118.1, 109.9, 102.3, 93.5, 93.1, 75.8, 64.6, 64.4, 44.4, 41.0, 36.2, 31.0, 30.6, 15.0 (x 2); HRMS (ESI-TOF): m/z: calculated for C30H36O7Na: 531.2359, found 531.2350 [M+Na^]. [α]25 D = - 24.1 (c 0.33, CHCl3).
Figure imgf000170_0003
53412 v2/DC JgQ. Compound 2-12Og: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.85-6.78 (m + s, 2H), 6.56 (d, J = 2.1 Hz, I H), 6.00 (d, J = 16.1 Hz, IH), 5.38-5.34 (m, 2H), 5.23 (s, 2H), 5.19 (s, 2H), 4.33 (t, J = 5.4 Hz, 2H), 3.75-3.70 (m, 6H), 2.45-2.41 (m, 2H), 2.19 (bs, 4H), 1.27-1.20 (m, 6H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 197.2, 168.1, 159.1, 155.8, 148.6, 134.5, 131.7, 129.8, 129.1 , 1 18.9, 109.9, 102.4, 93.6, 93.1 , 64.5, 64.5, 64.4, 45.4, 31.9, 31.1, 30.8, 15.0, 15.0; HRMS (ESI-TOF); m/z: calculated for C23H30O7Na: 441.1884, found 441.1888 [M+Nal.
Figure imgf000171_0001
(S)-2-121
Compound (S)-2-121 : Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): I O δ = 6.77-6.72 (m, IH), 6.57-6.47 (m, I H), 5.62-5.35 (m, 3H), 5.24-5.16 (m, 4H), 3.77-3.70 (m, 4H), 3.62-3.60 (m, 1.5H), 3.53-3.49 (m, 1.5H), 3.17-3.1 1 (m, 3H), 3.04-2.97 (m, IH), 2.57-2.44 (m, 2H), 2.36-1.99 (m, 6H), 1.37-1.33 (m, 3H), 1.28-1.21 (m, 8H); HRMS (ESI- TOF): m/z: calculated for C26H39O8NNa: 516.2568, found 516.2596 [M+Na+].
Figure imgf000171_0002
(R)-2-121a
15 Compound (7?)-2-121 a: Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.51 -7.42 (m, 2H), 7.38-7.31 (m, 3H), 6.73-6.70 (m, I H), 6.60-6.49 (m, I H), 6.45- 6.31 (m, I H), 5.73-5.39 (m, 2H), 5.23-5.00 (m, 4H), 3.75-3.69 (m, 2H), 3.56-3.34 (m, 6H), 3.19-3.09 (m, 3H), 2.66-2.08 (m, 8H), 1 .31 -1.19 (m, 5H), 1.10-1.04 (m, 3H); HRMS (ESI- TOF): m/z: calculated for C3|H4!O8NNa: 578.2724, found 578.2715 [M+Naτ].
53412 v2'DC ^ 70
Figure imgf000172_0001
(S)-2-121a
Compound (S)-2-l 2l a: Mixture of four diastereo isomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.51-7.42 (m, 2H), 7.38-7.29 (m, 3H), 6.73-6.70 (m, IH), 6.60-6.49 (m, IH), 6.45- 6.31 (m, I H), 5.73-5.42 (m, 2H), 5.25-5.01 (m, 4H), 3.76-3.69 (m, 2H), 3.62-3.34 (m, 6H), 3.20-3.09 (m, 3H), 2.66-2.50 (m, 2H), 2.22-2.12 (m, 4H), 1.72-1.66 (m, 2H), 1.31-1.19 (m, 5H), 1.10- 1.04 (m, 3H); HRMS (ESI-TOF): m/z: calculated for C3,H4iO8NNa: 578.2724, found 578.2720 [Λf+Na*].
Figure imgf000172_0002
(R)-2-121d
Compound (/?)-2-121d: Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ - 6.77 (s, I H), 6.52 (s, 0.5H), 6.46 (s, 0.5H), 5.59-5.37 (m, 2H), 5.21-5.18 (m, 4H), 5.09-4.92 (m, I H), 3.75-3.70 (m, 4H), 3.53-3.48 (m, 3H), 3.38-3.34 (m, IH), 3.19-3.10 (m, 3H), 2.65-2.47 (m, 3H), 2.29-2.04 (m, 6H), 1.89-1.72 (m, 2H), 1.31-1.20 (m, 6H), 1.06-0.96 (m, 6H); HRMS (ESI-TOF): m/r. calculated for C28H43O8NNa: 544.2881, found 544.2907 [M+Naτ].
Figure imgf000172_0003
2-121g
Compound 2-121g: Mixture of two diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 6.74-6.72 (m, IH), 6.54 (d, J - 1.8 Hz, 0.6H), 6.50 (d, J = 1.7 Hz, 0.4H), 5.52-5.41 (m,
53412 v2;DC \ η \ 2H), 5.21 (s, 2H), 5.19 (s, 2H), 4.69-4.65 (m, IH), 4.58-4.47 (m, IH), 3.74-3.68 (m, 4H), 3.55 (s, 3H), 3.13 (s, 3H), 2.97-2.94 (m, IH), 2.52-2.40 (m, 2H), 2.26-1.98 (m, 6H), 1.72-1.58 (m, 2H), 1.24-1.20 (m, 6H); HRMS (ESI-TOF): m/∑: calculated for C25H38O8N: 480.2567, found 480.2592 [M+YT).
[00353] General procedure for the EOM deprotection to generate compounds deprotected-2- 121 a-g, 2-85a-g and 2-103a-g: PS-TsOH (10.0 equiv., 3.2 mmol.g"1) was added to a solution of the corresponding compound 2-121a-g or 2-112a-g or 2-120a-g (1.0 equiv.) in MeOH (0.03 M) and the resulting suspension was shaken at 40 0C for 1 to 4 h. After this time, the reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-33 % EtOAc/cyclohexane gradient) afforded the corresponding compound deprotected-2-121a-g or 2-85a-g or 2-103a- g (> 90 %).
Figure imgf000173_0001
Deprotected (S)-2-121
Deprotected compound (_S)-2-l 21 : Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 12.90 (s, 0.5H), 12.83 (s, 0.5H), 12.12 (s, 0.5H), 12.02 (s, 0.5H), 6.92 (s, 0.5H), 6.84 (s, 0.5H), 6.83 (s, I H), 6.79 (s, 0.5H), 6.60 (s, I H), 6.54 (s, 0.5H), 5.60-5.29 (m, 4H), 5.17-4.99 (m, 2H), 4.14-3.99 (m, 2H), 2.98-2.72 (m, 12H), 2.60-1.92 (m, 16H), 1.31 (d, J = 6.4 Hz, 1.5H), 1.22 (d, J = 8.7 Hz, 1.5H), 1.13-1.02 (m, 7H); HRMS (ESI-TOF): m/z: calculated for C20H27O6NNa: 400.1851 , found 400.1731 [Λf+Naτ].
Figure imgf000173_0002
[ Deprotected (R)-2-121a
Deprotected compound (i?)-2-121 a: Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.90 (s, 0.25H), 11.08 (s, 0.5H), 10.98 (s, 0.25H), 7.38-7.29 (m, 5H),
53412 DC ] 72 6.39 (s, 0.25H), 6.33 (s, 0.25H), 6.29 (s, 1.25H), 6.26 (s, 0.25H), 6.05-5.95 (m, I H), 5.70- 5.52 (m, 2H), 4.18-4.03 (m, IH), 3.51 -3.49 (m, 3H), 3.16-3.14 (m, 3H), 2.75-2.62 (m, 2H), 2.36-2.29 (m, 2H), 2.12-1.96 (m, 4H), 1.81-1.73 (m, 2H), para-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C25H30O6N: 440.2068, found 440.2103 [M+H+].
Figure imgf000174_0001
Deprotected (S)-2-121a
Deprotected compound (S)-2-121a: Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.90 (s, 0.25H), 1 1.08 (s, 0.5H), 10.98 (s, 0.25H), 7.38-7.31 (m, 5H), 6.38 (s, 0.25H), 6.33 (s, 0.25H), 6.29 (s, 1.25H), 6.26 (s, 0.25H), 6.05-5.95 (m, IH), 5.71- 5.54 (m, 2H), 4.13-4.04 (m, I H), 3.53-3.50 (m, 3H), 3.19-3.14 (m, 3H), 2.78-2.63 (m, 2H), 2.33-2.29 (m, 2H), 2.16-2.04 (m, 4H), 1.81-1.68 (m, 2H), para-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C25H29O6NNa: 462.1887, found 462.2080 [M+Na+],
Figure imgf000174_0002
Deprotected (R)-2-121d
Deprotected compound (R)-2-\2\ ά: Mixture of four diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.54 (s, IH), 6.33 (d, J = 2.3 Hz, IH), 6.25 (s, IH), 5.53-5.51 (m, IH), 5.44-5.41 (m, IH), 5.1 1-5.08 (m, IH), 4.01 (d, J - 1 1.7 Hz, 2H), 3.45 (s, 3H), 3.1 1 (s, 3H), 2.83-2.73 (m, I H), 2.68-2.59 (m, IH), 2.27-2.20 (m, I H), 2.10-1.87 (m, 6H), 1.82-1.72 (m, IH), 1.01-0.94 (m, 6H), para-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C22H31O6NNa: 428.2044, found 428.2109 [M+Na+].
53412 v2/DC 173
Figure imgf000175_0001
Deprotected 2-121 g
Deprotected compound 2-121g: Mixture of two diastereoisomers; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 12.33 (s, 0.5H), 1 1.85 (s, 0.5H), 6.34-6.32 (m, IH), 6.25-6.22 (m, IH), 5.62-5.45 (m, 2H), 4.54-4.37 (m, IH), 4.29-4.21 (m, IH), 3.53-3.49 (m, 3H), 3.15-3.12 (m, 3.5H), 2.95-2.86 (m, 0.5H), 2.67-2.52 (m, 2H), 2.39-1.96 (m, 8H), pαrø-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C9H26O6N: 364.1755, found 364.1715 [M+H+].
Figure imgf000175_0002
Compound (5J-2-85: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.40 (s, IH), 6.83 (s, IH), 6.66- 6.61 (m, IH), 5.96 (bs, IH), 5.80 (d, J= 15.2 Hz, IH), 5.16-5.12 (m, IH), 4.99-4.91 (m, IH), 4.75-4.68 (m, I H), 4.26 (d, J = 17.5 Hz, IH), 4.13 (d, J = 17.5 Hz, I H), 2.52-2.45 (m, IH), 1.86-1.79 (m, 3H), 1.75-1.67 (m, I H), 1.54-1.49 (m, IH), 0.97 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 193.8, 169.8, 164.3, 156.8, 146.0, 137.1 , 131.9, 128.1, 126.2, 1 15.2, 107.6, 103.6, 72.4, 46.2, 36,3, 31.0, 30.8, 17.2. (-)-(25): [α]25 D = - 21.9 (c 0.62, CHCl3).
Figure imgf000175_0003
Compound (Λ)-2-85a: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.25 (bs, I H), 7.32-7.29 (m, 2H), 7.19-7.15 (m, 3H), 6.80 (s, I H), 6.81-6.75 (m, I H), 6.29-6.26 (m, IH), 5.92 (d, J = 15.8 Hz, IH), 5.80 (s, IH), 5.05-4.99 (m, IH), 4.81 -4.75 (m, I H), 4.56 (d, J = 17.6 Hz, IH), 4.12 (d, J = 17.5 Hz, IH), 2.73-2.66 (m, IH), 2.39-2.35 (m, IH), 1.86-1.76 (m, 2H), 1.64-1.49 (m,
53412 v2'DC 174 2H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 194.2, 169.8, 164.4, 156.8, 145.5, 138.3, 137.0, 132.9, 129.9 (x 2), 128.6, 127.3, 126.6 (x 2), 125.8, 1 15.2, 107.5, 103.6, 77.6, 46.6, 38.3, 31.0, 30.6; HRMS (ESI-TOF): m/z: calculated for C23H2 IO5ClNa: 435.0970, found 435.0914 [Λf+Na+]. [(X]25D = - 12.0 (c 0.55, CHCl3).
Figure imgf000176_0001
(S)-2-85a
Compound (5)-2-85a: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.20 (bs, I H), 7.15-7.09 (m, 3H), 6.75 (s, I H), 6.77-6.71 (m, IH), 6.25-6.22 (m, IH), 5.88 (d, J = 15.2 Hz, IH), 5.72 (s, IH), 5.00-4.95 (m, I H), 4.77-4.73 (m, I H), 4.52 (d, J = 17.6 Hz, I H), 4.08 (d, J = 17.5 Hz, I H), 2.68-2.62 (m, I H), 2.35-2.31 (m, I H), 1.81-1.78 (m, 2H), 1.56-1.49 (m, 2H), 2H masked by the solvent peak; 13C NMR (100 MHz, C6D6, 25 0C): δ = 194.2, 169.8, 164.4, 156.8, 145.5, 138.3, 137.0, 132.2, 129.9 (x 2), 128.5, 127.3, 126.5 (x 2), 125.8, 1 15.2, 107.5, 103.6, 77.5, 46.6, 38.2, 31.0, 30.6; HRMS (ESI-TOF): m/z: calculated for C23H2 IO5ClNa: 435.0970, found 435.0885 [M+Na÷]. [α]25 D = + 1 1.6 (c 0.51 , CHCl3).
Figure imgf000176_0002
(R)-2-85d
Compound (Λ)-2-85d: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.31 (s, IH), 6.83 (s, IH), 6.74-6.67 (m, IH), 5.84 (bs, IH), 5.82 (d, J = 15.8 Hz, IH), 5.03-4.95 (m, IH), 4.88-4.86 (m, I H), 4.76-4.70 (m, IH), 4.40 (d, J = 17.6 Hz, IH), 4.15 (d, J = 17.5 Hz, IH), 2.40-2.34 (m, IH), 2.22-2.18 (m, I H), 1.87-1.65 (m, 4H), 1.53-1.48 (m, IH), 0.92 (d, J = 6.4 Hz, 3H), 0.66 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 193.7, 164.2, 156.8, 145.8, 137.2, 131.8, 129.3, 126.3, 1 15.3, 107.9, 103.6, 82.1, 46.4, 33.3, 30.9, 30.7, 28.8, 20.1, 18.5, 18.3; HRMS (ESI-TOF): m/z: calculated for C20H23ClO5Na: 401.1126, found 401.1170 [M+Na+]. [CC]25 D = - 35.6 (c 0.52, CHCl3).
53412 v2/DC 175
Figure imgf000177_0001
Compound 2-85g: 1H NMR (400 MHz, CD3OD, 25 0C): δ = 6.74-6.68 (m, IH), 6.48 (s, IH), 5.86 (d, J = 15.2 Hz, I H), 5.31 -5.25 (m, 2H), 4.39 (t, J= 5.3 Hz, 2H), 4.27 (s, 2H), 2.43-2.40 (m, 2H), 2.25 (m, 4H), phenols not detected; i 3C NMR (100 MHz, CD3OD, 25 0C): δ = 196.9, 170.1, 161.9, 158.1 , 147.8, 135.9, 130.9, 130.2, 129.9, 115.2, 107.3, 102.4, 65.9, 46.2, 31.3, 30.9, 30.5; HRMS (ESI-TOF): m/z: calculated for Ci7Hi8O5Cl: 337.0837, found 337.0797 [M+H+].
Figure imgf000177_0002
Compound (S)-2-103: H NMR (400 MHz, CD3OD, 25 0C): δ = 6.78-6.71 (m, I H), 6.29 (d, J - 2.4 Hz, IH), 6.22 (d, J = 2.0 Hz, I H), 5.87 (d, J= 15.5 Hz, IH), 5.37-5.23 (m, 3H), 4.01 (d, J = 17.2 Hz, I H), 3.92 (d, J = 17.0 Hz, I H), 2.67-2.61 (m, IH), 2.29-2.15 (m, 5H), 1.31 (d, J = 6.4 Hz, 3H), phenols not detected; 13C NMR (100 MHz, CD3OD, 25 0C): δ = 198.5, 169.8, 164,2, 162.3, 148.4, 139.1 , 131.6, 129.6, 127.3, 1 1 1.7, 101.7, 72.0, 47.7, 36.8, 30.8, 30.7, 17.4, (one quartenary carbon is not detected); HRMS (ESI-TOF): m/z: calculated for C18H20O5Na: 339.1203, found 339.1 141 [M+N& ]. (-)-(2S): [α]25 D = - 45.1 (c 0.27, CHCl3).
Figure imgf000177_0003
Compound (/?)-2-103a: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.0 (bs, IH), 7.32-7.29 (m, 3H), 7.19-7.15 (m, 2H), 6.86-6.79 (m, IH), 6.51 (d, J = 2.4 Hz, IH), 6.27-6.25 (m, IH), 6.1 1 (d, J = 2.4 Hz, IH), 6.02 (d, J = 15.8 Hz, IH), 5.49 (s, IH), 5.17-5.10 (m, I H), 4.97-4.90 (m, IH), 4.40 (d, J = 16.4 Hz, IH), 3.97 (d, J = 17.2 Hz, IH), 2.83-2.76 (m, IH), 2.45-2.38 (m,
53412 v2/DC 176 IH), 1.89-1.78 (m, 2H), 1.67-1.58 (m, 2H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 196.5, 169.6, 166.1 , 161.3, 146.0, 140.5, 138.8, 132.1 , 130.0, 128.6 (x 2), 127.3, 126.6 (x 2), 126.3, 1 12.2, 105.9, 103.0, 77.1 , 48.6, 38.4, 30.9, 30.3; HRMS (ESI-TOF): m/z: calculated for C23H22O5Na: 401.1359, found 401.1271 [Λf+Na*]. [α]25 D = - 10.3 (c 0.25, CHCl3).
Figure imgf000178_0001
Compound (S)-2-103a: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.0 (bs, IH), 7.27-7.21 (m, 3H), 7.17-7.13 (m. 2H), 6.87-6.79 (m, 1 H), 6.55 (d, J = 2.3 Hz, 1 H), 6.29-6.26 (m, 1 H), 6.16 (d, J = 2.3 Hz, I H), 6.03 (d, J = 15.8 Hz, IH), 5.74 (s, I H), 5.18-5.12 (m, I H), 4.98-4.91 (m, I H), 4.41 (d, J = 15.8 Hz, I H), 3.99 (d, J = 16.9 Hz, IH), 2.84-2.77 (m, IH), 2.46-2.43 (m, I H), 1.85-1.79 (m, 2H), 1.70-1.58 (m, 2H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 196.9, 169.6, 166.2, 161.5, 146.3. 140.5, 138.9, 132.1 , 130.0, 128.6 (x 2), 127.3, 126.6 (x 2), 126.3, 1 12.2, 105.8, 103.0, 77.1 , 48.6, 38.4, 30.9, 30.4; HRMS (ESI-TOF): m/z: calculated for C23H22O5Na: 401.1359, found 401.1264 [M+Naτ]. [α]25 D = + 1 1.9 (c θ.51, CHCl3).
Figure imgf000178_0002
Compound (i?)-2-103d: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.10 (s, IH), 6.79 (dt, J = 15.2, 7.6 Hz, I H), 6.59 (d, J = 2.4 Hz, IH), 6.22 (d, J = 2.3 Hz, IH), 5.96 (d, J = 15.8 Hz, IH), 5.88 (bs, IH), 5.13-5.05 (m, IH), 4.92-4.85 (m, 2H), 4.27 (d, J= 15.8 Hz, IH), 4.03 (d, J = 15.8 Hz, I H), 2.50-2.44 (m, I H), 2.24-2.20 (m, IH), 1.96-1.71 (m, 4H), 1.63-1.56 (m, IH), 0.91 (d, J = 6.3 Hz, 3H), 0.71 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 196.5, 169.7, 166.0, 161.4, 146.8, 140.8, 131.7, 129.5, 126.9, 1 12.3, 106.0, 103.0, 81.3, 48.5, 33.7, 30.9, 30.4, 29.6, 19.7, 18.4; HRMS (ESI-TOF): m/z: calculated for C20H24O5Na: 367.1516, found 367.1424 [M+Naτ]. [α]25 D = - 31.9 (c 0.50, CHCl3).
53412 v2 DC 177
Figure imgf000179_0001
Compound (/?)-2-103e: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 12.43 (s, IH), 6.74 (d, J = 1.7 Hz, IH), 6.73-6.65 (m, I H), 6.48 (d, J= 1.7 Hz, IH), 5.92 (d, J= 15.8 Hz, IH), 5.12-5.00 (m, 2H), 4.91-4.80 (m, IH), 4.19 (d, J = 17.0 Hz, IH), 3.84 (d, J = 16.4 Hz, IH), 2.77 (m, I H), 2.64-2.57 (m, IH), 2.01-1.97 (m, IH), 1.89-1.70 (m, 3H), 1.61-1.56 (m, 2H), 1.30-1.21 (m, 2H), 0.90 (t, J = 6.7 Hz, 3H), /κ*rα-ρhenol not detected; 13C NMR (100 MHz, CDCl3, 25 0C): δ = 197.5, 169.9, 165.6, 160.6, 147.5, 140.2, 131.9, 129.5, 127.0, 1 12.8, 106.1 , 102.9, 76.2, 48.7, 35.7, 34.3, 31.1, 29.7, 19.4, 13.8; HRMS (ESI-TOF): m/z: calculated for C20H25O5: 345.1697, found 345.1739 [M+H+]. [α]25 D = + 21.6 (c 0.36, CHCl3).
Figure imgf000179_0002
Compound (Λ)-2-103f: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 12.31 (s, I H), 7.19-7.13 (m, 5H), 6.80-6.72 (m, I H), 6.53 (d, J = 1.8 Hz, IH), 6.05 (s, I H), 5.89 (d, J = 15.8 Hz, I H), 5.47-5.44 (m, I H), 5.1 1 -5.05 (m, I H), 4.85-4.81 (m, I H), 4.10 (d, J= 17.0 Hz, I H), 3.62 (d, J = 17.0 Hz, I H), 2.89-2.84 (m, I H), 2.67-2.60 (m, 2H), 2.08-2.04 (m, I H), 1.90-1.69 (m, 3H), 1.52-1.44 (m, I H), pαrα-phenol not detected; 13C NMR (100 MHz, CDCl3, 25 0C): δ = 197.7, 169.9, 165.7, 160.7, 147.4, 140.2, 137.2, 132.2, 129.5, 128.8 (x 2), 128.7 (x 3), 126.8, 1 12.4, 105.9, 102.9, 48.9, 38.5, 35.3, 31.1 (x 2), 29.7; HRMS (ESI-TOF): m/z: calculated for C24H25O5 : 393.1697, found 393.1765 [M+H+]. [α]25 D = + 25.4 (c 0.41, CHCl3).
Figure imgf000179_0003
53412 v2 'DC 178 Compound 2-103g: 1H NMR (400 MHz, CD3OD, 25 0C): δ = 6.75-6.69 (m, IH), 6.29 (s, IH), 6.29 (d, J = 2.3 Hz, IH), 5.90 (d, J - 15.8 Hz, IH), 5.30-5.28 (m, 2H), 4.39 (t, J = 5.2 Hz, 2H), 4.02 (s, 2H), 2.45-2.41 (m, 2H), 2.28-2.24 (m, 4H), phenols not detected; 13C NMR (100 MHz, CD3OD, 25 0C): δ = 198.4, 170.6, 164.9, 162.5, 148.1 , 139.2, 130.7, 130.6, 130.2, 1 12.2, 105.0, 101.6, 65.7, 47.7, 31.4, 30.9, 30.5; HRMS (ESI-TOF): m/z: calculated for C πH 19O5 : 303.1227, found 303.1 179 [M+H+].
[00354] General procedure for the synthesis of compounds 2-141 : BER-resin (Borohydride on Amberlite, 1.0 equiv., 2.5 mmol.g'1) was added to a solution of corresponding compound 2-112a-g or 2-120a-g (1.0 equiv.) in MeOH (0.03 M) at 0 0C and the reaction was stirred for 12 h. The reaction mixture was then filtered and concentrated under reduced pressure. Purification by flash chromatography (silica gel, 0-20 % EtOAc/cyclohexane gradient) afforded 2-141 (~60 % yield) as a mixture of two diastereoisomers 1 : 1.
Figure imgf000180_0001
[00355] Selected example of compounds 2-141 : 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.05 (s, I H), 6.99 (s, I H), 5.64-5.57 (m, 2H), 5.54-5.53 (m, 2H), 5.49-5.35 (m, 7H), 5.31-5.28 (m, 4H), 5.24-5.16 (m, 4H), 5.13-5.08 (m, I H), 4.68 (m, IH), 4.56 (m, IH), 3.81-3.69 (m, 8H), 3.25 (dd, J = 13.9, 8.0 Hz, IH), 3.19 (dd, J = 13.7, 4.8 Hz, IH), 3.1 1 (dd, J= 13.5, 10.1 Hz, I H), 2.90 (dd, J = 13.9, 5.1 Hz, IH), 2.35 (m, 9H), 2.09-1.95 (m, IH), 1.80-1.70 (m, 2H), 1.39 (d, J= 2.9 Hz, 3H), 1 .37 (d, J = 3.2 Hz, 3H5), 1.24 (2 x q, J= 6.9, 5.0 Hz, 12H); HRMS (ESI-TOF): m/z: calculated for C24H33ClO7Na: 491.1807, found 491.1729 [Λ/+Na+].
General procedure for the synthesis of compounds 2-142: PS-TsOH (10.0 equiv., 3.2 mmol.g" ') was added to a solution of the corresponding compound 2-141 (1.0 equiv.) in MeOH (0.02 M) and the suspension was shaken at 40 0C for 4 h. The reaction mixture was then filtered and the methanolic solution concentrated under reduced pressure. Purification by preparative TLC (silica gel, 25 % EtOAc/cyclohexane) afforded 2-142 (-90 % yield) as a mixture of two diastereoisomers 1 : 1.
53412 v2.'DC 1 79
Figure imgf000181_0001
[00356] Selected example of compounds 2-142: 1H NMR (400 MHz, (CD3)2CO, 25 0C): δ =12.30 (s, 2H), 1 1.43 (s, 2H), 6.75 (s, 2H), 6.00 (bdd, J= 6.4, 6.2 Hz, IH), 5.97 (bdd, J= 6.4, 6.2 Hz, IH), 5.97 (bd, J= 6.7 Hz, IH), 5.77 (bd, J= 6.7 Hz, IH), 5.57-5.48 (m, 4H), 5.18- 5.14 (m, 2H), 3.38-3.28 (m, 3H), 3.02 (dd, J= 16.1, 10.5 Hz, IH), 2.41-2.09 (m, 12H), 1.11 (d, J= 6.2 Hz, 6H), alcohols not detected; HRMS (ESI-TOF): m/z: calculated for C18H21ClO5Na: 375.0970, found 375.1029 [M+Na+].
[00357] General procedure for the synthesis of compounds 2-143: Ac2O (1.2 equiv.), morpholinomethyl polystyrene (1.2 equiv., 3.2 mmol.g"1) and DMAP (0.05 equiv.) were added to a solution of the corresponding compound 2-141 (1.0 equiv.) in DMF (0.02 M) at 23 0C and the mixture was stirred for 30 min, followed by TLC until consumption of the starting material. The resin was then filtered and the organic phase was concentrated under reduced pressure. Purification by preparative TLC (silica gel, 20 % EtOAc/cyclohexane) afforded corresponding compound 2-143 (~80 % yield) as a mixture of two diastereoisomers 1 : 1.
Figure imgf000181_0002
[00358] Selected example of compounds 2-143: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.04 (s, IH), 7.01 (s, I H), 5.86 (dd, J= 15.0, 6.9 Hz, IH), 5.67 (dd, J= 12.4, 6.2 Hz, IH), 5.60-5.54 (m, 4H), 5.48 (dd, J= 7.2, 7.2 Hz, I H), 5.41-5.34 (m, 3H), 5.32-5.30 (m, 4H), 5.28- 5.23 (m, 2H), 5.21 (dd, J = 1 1.0, 6.7 Hz, 2H), 5.17 (dd, J= 1 1.8, 6.9 Hz, 2H), 3.81-3.69 (m, 8H), 3.43 (dd, J= 14.2, 7.5 Hz, IH), 3.23-3.15 (m, 2H), 2.85 (dd, J= 13.9, 5.4 Hz, IH), 2.30- 2.17 (m, 8H), 2.12 (s, 3H), 2.06 (s, 3H), 2.00-1.95 (m, 4H), 1.39 (2 x d, J= 5.6 Hz, 6H), 1.24 (m, 12H); HRMS (ESI-TOF): m/z: calculated for C26H35ClO8Na: 533.1913, found 533.1864 [M+Na"]. [00359] General procedure for the synthesis of compounds 2-144: PS-TsOH (10.0 equiv., 3.2 mmol.g"1) was added to a solution of corresponding compound 2-143 (1.0 equiv.) in MeOH (0.02 M) and the suspension was shaken at 40 0C for 4 h. After this time, the reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. Purification by
53412 \2 DC ] gθ preparative TLC (silica gel, 20 % EtOAc/cyclohexane) afforded compound 2-144 (-60 % yield).
Figure imgf000182_0001
[00360] Selected example of compounds 2-144: Mixture of diastereoisomers 2: 1 ; H NMR (400 MHz, CDCl3, 25 0C): δ = 12.60 (s, IH), 12.12 (s, 0.5H), 6.93 (d, J- 8.7 Hz, 0.5H), 6.66 (s, IH), 6.64 (s, 0.5H), 6.62-6.60 (m, I H), 6.10-6.05 (m, 3H), 5.47-5.33 (m, 6H), 2.60-2.53 (m, 1.5H), 2.26-2.02 (m, 7.5H), 1.44 (d, J - 6.2 Hz, 1.5H), 1.43 (d, J = 6.4 Hz, 3H), para- phenol not detected; HRMS (ESI-TOF): m/z: calculated for Ci8Hi9ClO4Na: 357.0864, found 357.0898 [M+Na*].
[00361 ] General procedure for the preparation of compounds 2-145: PS-TsOH (10.0 equiv.) was added to a solution of corresponding compound 2-85a-g (1.0 equiv.) in methanol (0.03 M) and the suspension was stirred for 15 h at 4O0C. The reaction was then filtered and the resin washed several times with CH2Cl2. Concentration under reduced pressure followed by purification on preparative TLC (silica gel, 50 % hexane/EtOAc) afforded desired compound 2-145 as a mixture of diastereoisomers 2:1.
Figure imgf000182_0002
[00362] Selected example of compounds 2-145: 1H NMR (400 MHz, C6D6, 25 0C): δ = 12.28 (s, 0.4H), 1 1.91 (s, 0.6H), 7.21-7.1 1 (m, 5H), 6.62 (s, IH), 6.03-6.01 (m, IH), 5.58 (bs, I H), 5.38-5.33 (m, IH), 5.27-5.20 (m, IH), 4.76 (d, J= 17.5 Hz, 0.6H), 4.02 (d, J= 17.0 Hz, 0.4H), 4.18 (d, J= 18.1 Hz, 0.6H), 4.09 (d, J= 17.0 Hz, 0.4H), 3.87 (bs, 0.4H), 3.81 (bs, 0.6H), 3.15 (s, 1.8H), 3.12 (s, 1.2H), 2.83-2.78 (m, I H), 2.45-2.30 (m, 2H), 2.18-2.16 (m, IH), 2.02-1.97 (m, 2H), 1.79-1.72 (m, 2H); HRMS (ESI-TOF): m/z: calculated for C24H25O6ClNa: 467.1232, found 467.1366 [M+Na+]. [00363] General procedure for the synthesis of compounds 2-146: (Polystyrylmethyl)trimethyl-ammonium cyanoborohydride (2.0 equiv., 3.5 mmol.g"1) was added to a solution of corresponding compound 2-85a-g or 2-103a-g (1.0 equiv.) in CH2Cl2/Ac0H 10: 1 (0.08 M) at 23 0C and the reaction was monitored by TLC until
53412 v2, DC | g ] consumption of the starting material (4 h). The resin was then filtered and the organic phase was concentrated under reduced pressure. Purification by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) afforded compound 2-146 (50-60 % yield).
Figure imgf000183_0001
[00364] Selected example of compounds 2-146: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.75 (s, I H), 6.65 (s, I H), 5.48 (m, 2H), 5.49 (ddt, J- 6.1, 3.5, 2.9 Hz, IH), 4.53 (d, J= 17.5 Hz, I H). 4.04 (d, J = 17.7 Hz, I H), 2.61 -2.54 (m, 2H), 2.48-2.28 (m, 3H), 2.19-2.14 (m, IH), 2.08-1.99 (m, I H), 1 .72-1 .61 (m, 3H), 1.41 (d, J = 6.4 Hz, 3H), /κzra-phenol not detected; HRMS (ESI-TOF): m/z: calculated for Ci8H2 IClO5Na: 375.0970, found 375.1050 [Λf+Na*]. [00365] General procedure for the synthesis of compounds 2-147: The corresponding alcohol R2OH (2.0 equiv.), triphenylphosphine (2.0 equiv.) and PS-DEAD (2.0 equiv., 1.3 mmol.g'1) were added to a solution of corresponding compound 2-85a-g or 2-103a-g (1 .0 equiv.) in THF (0.05 M) in a sequential manner. The reaction mixture was shaken at room temperature for 8 h, and then the resin was filtered and the filtrates were directly purified by preparative TLC (silica gel, 10 % EtOAc/cyclohexane) to afford a mixture of compound 2-147 along with the bis-alkylated product (78 % yield).
Figure imgf000183_0002
[00366] Selected example of compounds 2-147: Mixture with the corresponding bis-allylated compound 1 : 1 ; 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.83 (s, IH), 6.82 (ddd, J= 15.7, 8.2, 4.6 Hz, I H), 6.72-6.65 (m, IH), 6.46 (s, I H), 6.41 (s, IH), 6.09-5.98 (m, 3H), 5.82 (d, J = 15.7 Hz, I H), 5.46-5.16 (m, 8H), 4.57-4.54 (m, 3H), 4.51-4.49 (m, 3H), 4.19 (d, J= 17.5 Hz, I H), 4.1 1 (d, J = 14.6 Hz, I H), 3.78 (d, J= 17.0 Hz, IH), 3.51 (d, J= 14.2 Hz, I H), 2.76-2.69 (m, I H), 2.38-2.05 (m, H H), 1.42 (d, J= 6.2 Hz, 3H), 1.35 (d, J= 6.3 Hz, 3H); mono- allylated compound: HRMS (ESI-TOF): m/z: calculated for C2IH23ClO5Na: 413.1 132, found 413.1 103 [M+Na"]; bis-allylated compound: HRMS (ESI-TOF): m/z: calculated for C24H27ClO5Na: 453.1449, found 453.1422 [M+Na*].
[00367] General procedure for the synthesis of compounds 2-148: TBD-methyl polystyrene (2.0 equiv., 2.9 mmol.g"1) and the corresponding alkyl bromide or chloride (BrCH2COO1Bu,
53412 v2 'DC ] g2 EOMCl, 0.9 equiv.) were added to a solution of the corresponding compound 2-85a-g or 2- 103a-g (1.0 equiv.) in CH2Cl2 (0.05 M) at 23 0C and the mixture was shaken for 3 h. The resin was then filtered and the filtrates were concentrated under reduced pressure. Purification by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) afforded corresponding compound 2-148 (> 90 % yield).
Figure imgf000184_0001
[00368] Selected examples of compounds 2-148: 1H NMR (400 MHz, CDCl3, 25 0C): δ - 1 1.84 (s, IH), 6.69 (m, I H), 6.41 (s, IH), 5.76 (d, J= 15.0 Hz, IH), 5.43 (m, IH), 5.26 (ddd, J= 15.0, 9.1 , 4.8 Hz, I H), 5.18-5.1 1 (m, IH), 4.65 (s, 2H), 4.33 (d, J= 17.7 Hz, IH), 4.16 (d, J= 17.5 Hz, I H), 2.65-2.58 (m, I H), 2.37-2.34 (m, 2H), 2.25-2.21 (m, IH), 2.12-2.01 (m, 2H), 1.53 (s, 9H), 1.34 (d, J = 6.5 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C24H29ClO7Na: 487.1494, found 487.1498 [Λf+Naτ].
Figure imgf000184_0002
1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.76 (s, IH), 6.86 (s, IH), 6.70 (dt, J = 14.9, 7.3 Hz, IH), 5.77 (d, J = 15.8 Hz, IH), 5.46-5.42 (m, IH), 5.37 (s, 2H), 5.30-5.19 (m, 2H), 4.34 (d, J = 17.6 Hz, I H), 4.16 (d, J = 18.1 Hz, IH), 3.80 (q, J = 7.0 Hz, 2H), 2.66-2.59 (m, IH), 2.37- 2.34 (m, 2H), 2.26-2.21 (m, IH), 2.13-2.06 (m, 2H), 1.34 (d, J = 6.4 Hz, 3H), 1.27 (t, J= 7.0 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C2IH25O6ClNa: 431.1237, found 431.1257 [Λf+Nax].
[00369] General procedure for the synthesis of compounds 2-149: OsO4 (0.1 equiv.) and
NMO (1.0 equiv.) were added to a solution of compound 2-85a-g or 2-103a-g (1.0 equiv.) in acetone/H2O 10: 1 (0,05 M) at 23 0C and the mixture was stirred for 1 h. The crude mixture was filtered through a pad of silica, concentrated and purified by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) to afford 2-149 as a mixture of two diastereoisomers 1 : 1 (> 70 % yield).
53412 v2/DC J §3
Figure imgf000185_0001
[00370] Selected example of compounds 2-149: 1H NMR (400 MHz, CD3OD, 25 0C): δ - 7.19 (m, IH), 6.89-6.81 (m, IH), 6.52 (s, IH), 6.47 (s, IH), 6.20 (d, J= 16.1 Hz, IH), 6.04 (d, J = 15.6 Hz, IH), 5.54-5.49 (m, IH), 5.43-5.36 (m, IH), 4.50 (d, J= 17.7 Hz, I H), 4.46 (d, J = 17.7 Hz, IH), 4.39 (d, J= 17.2 Hz, IH), 4.07 (d, J= 17.2 Hz, IH), 3.80-3.64 (m, 2H), 3.51- 3.46 (m, 2H), 2.62-2.58 (m, 2H), 2.39-2.30 (m, 2H), 2.27-2.18 (m, 2H), 2.08-1.98 (m, 2H), 2.00-1.85 (m, 4H), 1.44 (d, J= 6.4 Hz, 6H), phenols and alcohols not detected; HRMS (ESI- TOF): m/z: calculated for C8H2 I ClO7Na: 407.0868, found 407.1031 [M+Naτ]. [00371] General procedure for the synthesis of compounds 2-150: Freshly prepared DMDO (1.2 equiv., 0.04 M in acetone) was added to a solution of compound 2-85a-g or 2-103a-g (1.0 equiv.) in CH3CN (0.03 M) at 0 0C and the mixture was stirred for 30 min. After evaporation of the solvents under reduced pressure, purification by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) afforded epoxides 2-150 (> 90 % yield) as a mixture of two diastereoisomers (1 : 1 to 3: 1 ).
Figure imgf000185_0002
[00372] Selected examples of compounds 2-150: 1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.85 (s, I H), 6.94-6.87 (m, 2H), 6.69 (s, IH), 6.65 (s, I H), 6.24 (bd, J= 15.2 Hz, 2H), 6.12 (d, J= 15.8 Hz, IH), 5.41 -5.37 (m, IH), 5.33-5.30 (m, IH), 4.54 (bd, J= 18.1 Hz, 2H), 4.52- 4.48 (m, IH), 4.40-4.34 (m, IH), 4.27 (d, J= 17.5 Hz, IH), 2.78-2.72 (m, 2H), 2.58-2.55 (m, 4H), 2.47-2.28 (m, 5H), 2.07 (m, 2H), 1.92-1.86 (m, 3H), 1.51 (d, J= 6.4 Hz, 3H), 1.35 (d, J = 6.4 Hz, 3H), pαra-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C18H19O6ClNa: 389.0762, found 389.0844 [M+Na*].
Figure imgf000185_0003
53412 v2 'DC 184 1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.80 (2 x s, 2H), 7.43-7.18 (m, 10H), 7.03-6.95 (m, 2H), 6.69 (s, I H), 6.61 (s, IH), 6.30 (d, J = 16.4 Hz, IH), 6.21 (d, J = 15.8 Hz, IH), 6.15-6.10 (m, I H), 6.03 (d, J = 1 1.1 Hz, IH), 4.84 (2 x d, J = 18.1 Hz, 2H), 4.41 (2 x d, J = 17.6 Hz, 2H), 2.68-2.60 (m, 4H), 2.41 -2.27 (m, 8H), 1.83-1.76 (m, 4H), para-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C23H2)O6ClNa: 451.0919, found 451.1028 [M+Na"].
Figure imgf000186_0001
1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.56 (2 x s, 2H), 6.92-6.82 (m, 2H), 6.71 (s, IH), 6.67 (s, I H), 6.20 (m, 3H), 6.06 (d, J = 15.8 Hz, IH), 5.1 1 (bs, IH), 5.94 (m, IH), 4.46 (2 x d, J = 18.1 Hz, 2H), 4.20 (2 x d, J = 18.1 Hz, 2H), 2.72-2.70 (m, 2H), 2.53-2.48 (m, 4H), 2.38- 2.35 (m, 3H), 2.25-2.13 (m, 5H), 1.84-1.77 (m, 2H), 1.05-1.01 (m, 6H), 0.91-0.88 (m, 3H), 0.86-0.84 (m, 3H), /rørø-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C2OH23O6ClNa: 417, 1075, found 417.1 128 [M+Na+].
Figure imgf000186_0002
1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.98 (s, IH), 6.91-6.83 (m, IH), 6.43 (d, J = 2.3 Hz, IH), 6.24 (d, J = 2.4 Hz, IH), 6.1 1 (d, J = 15.8 Hz, IH), 5.35 (bs, IH), 5.29 (m, IH), 4.52 (d, J = 17.5 Hz, IH), 3.63 (d, J = 17.5 Hz, IH), 2.77 (m, 2H), 2.57-2.52 (m, 2H), 2.46-2.27 (m, 2H), 2.14-2.10 (m, IH), 1.93-1.88 (m, IH), 1.48 (d, J = 6.4 Hz, 3H); other isomer; 1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.67 (s, IH), 6.89-6.83 (m, IH), 6.40 (d, J= 2.4 Hz, IH), 6.24 (d, J = 2.9 Hz, IH), 6.21 (d, J = 16.4 Hz, IH), 5.37 (bs, IH), 5.22 (m, IH), 4.20 (d, J = 17.0 Hz, I H), 4.06 (d, J= 17.0 Hz, IH), 2.74 (m, 2H), 2.57-2.20 (m, 4H), 1.80-1.76 (m, IH), 1.68- 1.60 (m, I H), 1.37 (d, J = 6.4 Hz, 3H), para-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C18H20O6Na: 355.1 152, found 355.1249 [M+Na+].
Figure imgf000186_0003
53412 v2 DC 185 1H NMR (400 MHz, C6D6, 250C): δ = 11.98 (s, IH), 6.91-6.83 (m, IH), 6.43 (d, J = 2.3 Hz, IH), 6.24 (d, J= 2.4 Hz, IH), 6.11 (d,J= 15.8 Hz, IH), 5.35 (bs, IH), 5.29 (m, IH), 4.52 (d, J= 17.5 Hz, IH), 3.63 (d, J= 17.5 Hz, IH), 2.77 (bs, IH), 2.57-2.52 (m, 2H), 2.46-2.27 (m, 2H), 2.14-2.10 (m, IH), 1.93-1.88 (m, IH), 1.48 (d, J= 6.4 Hz, 3H).
Figure imgf000187_0001
Major isomer: 1H NMR (400 MHz, C6D6, 250C): δ = 11.90 (s, IH), 7.41-7.23 (m, 5H), 6.95- 6.89 (m, 1 H), 6.42 (d, J = 2.8 Hz, 1 H), 6.27 (d, J = 2.9 Hz, 1 H), 6.20 (d, J = 15.8 Hz, 1 H), 6.13 (d,J=4.1 Hz, IH), 5.51 (m, IH), 4.79 (d, J = 17.5 Hz, IH), 3.79 (d, J= 17.0Hz, IH), 2.68-2.55 (m, 3H), 2.44-2.25 (m, 4H), para-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C23H22O6Na: 417.1309, found 417.1399 [M+Naτ].
Figure imgf000187_0002
1H NMR (400 MHz, C6D6, 250C): 6=11.77 (s, IH), 6.92-6.82 (m, IH), 6.44 (d, J= 2.3 Hz, IH), 6.28 (d, J =2.4 Hz, IH), 6.09 (d, J= 15.8Hz, IH), 5.40 (bs, IH), 4.92 (m, IH), 4.50 (d, J= 17.5 Hz, IH), 3.61 (d, J= 17.5 Hz, IH), 2.72-2.70 (m, IH), 2.56-2.45 (m, 2H), 2.38-2.15 (m, 4H), 1.91-1.85 (m, IH), 1.05-1.01 (m, 6H), para-phenol not detected; other isomer: 1H NMR (400 MHz, C6D6, 250C): δ = 11.55 (s, IH), 6.86-6.79 (m, IH), 6.42 (s, IH), 6.29 (s, IH), 6.20 (d, J= 15.8Hz, IH), 5.40 (m, IH), 5.16 (m, IH), 4.14 (s, IH), 4.12 (s, IH), 2.72- 2.70 (m, IH), 2.53-2.37 (m, 4H), 2.18-2.10 (m, 2H), 1.92-1.86 (m, IH), 0.91-0.85 (m, 6H), pαrα-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C20H24O6Na: 383.1465, found 383.1574 [M+Na~],
Figure imgf000187_0003
53412 v2,DC 186 1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.87 (s, IH), 6.90-6.82 (m, IH), 6.43 (s, I H), 6.26 (s, I H), 6.10 (d, J = 15.2 Hz, IH), 5.31 (bs, I H), 5.18 (bs, I H), 4.46 (d, J = 17.5 Hz, IH), 3.60 (d, J = 17.6 Hz, I H), 2.74 (bs, I H), 2.57-2.38 (m, 3H), 2.32-2.22 (m, IH), 2.08-1.82 (m, 2H), 1.73-1.67 (m, IH), 1.42-1.37 (m, 2H), 1.33-1.28 (m, 2H), 1.01 (t, J = 7.3 Hz, 3H); other isomer: 1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.72 (s, IH), 6.86-6.80 (m, IH), 6.41 (s, IH), 6.27 (s, IH), 6.21 (d, J = 16.4 Hz, IH), 5.37 (m, IH), 5.22 (m, IH), 4.25 (d, J= 16.4 Hz, IH), 3.96 (d, J = 16.4 Hz, IH), 2.74 (bs, IH), 2.60-2.37 (m, 4H), 1.87-1.78 (m, 2H), 1.70-1.58 (m, 3H), 1.38-1.22 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C20H24O6Na: 383.1465, found 383.1492 [M+Na+].
Figure imgf000188_0001
Major isomer: 1H NMR (400 MHz, C6D6, 25 0C): δ = 11.94 (s, IH), 7.36-7.28 (m, 5H), 6.95- 6.88 (m, IH), 6.42 (s, IH), 6.22 (s, IH), 6.1 1 (d, J = 15.8 Hz, IH), 5.47 (m, IH), 5.41 (bs, I H), 4.43 (d, J = 17.5 Hz, IH), 3.56 (d, J = 17.6 Hz, IH), 3.19 (dd, J = 13.7, 6.0 Hz, IH), 3.03 (dd, J = 13.7, 7.9 Hz, I H), 2.87 (bs, IH), 2.70-2.28 (m, 4H), 2.03-1.93 (m, 2H), para- phenol not detected; HRMS (ESI-TOF): m/z: calculated for C24H24O6Na: 431.1465, found 431.1578 [M+Naτ].
[00373] General procedure for the preparation of macrocycles 2-151 : HClconc (20.0 equiv.) was added to a solution of compound 2-120 (1.0 equiv.) in dioxane (0.05 M) at 23 0C, and the mixture was stirred for 3 h. After this time, the reaction was filtered through a pad of silica, the solvents evaporated under reduced pressure, and purification by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) afforded compound 2-151 (> 75 % yield) as a mixture of two diastereoisomers 1 : 1.
Figure imgf000188_0002
[00374] Selected examples of compounds 2-151 : 1H NMR (400 MHz, CDCl3, 25 0C): δ = 12.1 1 (s, I H), 1 1.78 (s, I H), 6.51 (s, I H), 6.43 (s, I H), 6.41 (d, J = 2.4 Hz, IH), 6.37 (d, J = 2.7 Hz, IH), 6.21 (d, J= 2.4 Hz, IH), 6.1 1 (d, J= 2.4 Hz, I H), 5.59-5.51 (m, 3H), 5.40-5.32
53412 v2'DC 137 (m, 3H), 4.54 (d, J= 17.2 Hz, IH), 4.42 (d, J= 17.2 Hz, IH), 3.60 (d, J= 17.2 Hz, IH), 3.45 (d, J= 17.0 Hz, IH), 3.28 (dd, J= 18.5, 9.4 Hz, IH), 3.11 (dd, J= 13.7, 6.2 Hz, IH), 3.07 (dd, J= 13.4, 4.6 Hz, IH), 2.76 (dd, J= 19.0, 6.2 Hz, IH), 2.62 (ddd, J= 15.5, 8.8, 4.0 Hz, IH), 2.54 (ddd, J= 15.3, 6.2, 3.2 Hz, I H), 2.40-2.26 (m, 4H), 2.25-2.13 (m, 4H), 2.03-1.91 (m, 2H), 1.42 (d, J = 6.4 Hz, 3H), 1.40 (d, J= 6.4 Hz, 3H), /?αra-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C18H21ClO5Na: 375.0970, found 375.0928 [M+Na+].
Figure imgf000189_0001
1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.76 (s, 0.5H), 1 1.36 (s, 0.5H), 7.40-7.29 (m, 5H), 6.65 (s, 0.5H), 6.62 (s, 0.5H), 6.18 (t, J = 5.8 Hz, I H), 6.14 (s, 0.5H), 6.12 (s, 0.5H), 5.67- 5.62 (m, I H), 5.55-5.49 (m, I H), 4.93 (d, J = 18.1 Hz, 0.5H), 4.80 (d, J = 17.1 Hz, 0.5H), 4.58-4.56 (m, I H), 4.38 (d, J = 18.1 Hz, 0.5H), 4.18 (d, J = 17.1 Hz, 0.5H), 3.33-3.27 (m, I H), 3.10 (dd, J = 18.4, 3.8 Hz, 0.5H), 2.84-2.68 (m, 2.5H), 2.42-2.32 (m, 2H), 2.23-2.17 (m, I H), 2.13-2.04 (m, I H), pαrα-phenol not detected; HRMS (ESI-TOF): m/z: calculated for C23H22O5Cl2Na: 471.0737, found 471.0754 [M+Na"].
[00375] Elimination of β-Cl from compound 2-151 : PS-TBD (51 mg, 2.6 mmol.g"1) was added to a solution of compound 2-151 (95 mg, 270 μmol) in CH2Cl2 (5 mL) at 23 0C, and the mixture was stirred for 8 h. After this time, the reaction was filtered, the solvents were evaporated under reduced pressure, and the remaining residue was purified by flash chromatography (silica gel, 0-30 % EtOAc/cyclohexane gradient) to afford 2-103 (84 mg, 98 %).
[00376] General procedure for the synthesis of compounds 2-152: Compound 2-12Oa or 2- 112a (1.0 equiv.) was dissolved in a 1 :5 mixture of TFA/CH2CI2 and stirred for 2 h at room temperature. Evaporation of the solvents; followed by flash chromatography (silica gel, 0-50 % Et2O/hexane), afforded compound 2-152 (-70 % yield).
Figure imgf000189_0002
53412 v2/DC [00377] Selected example of compounds 2-152: 1H NMR (400 MHz, C6D6, 25 0C): δ = 1 1.74 (s, I H), 7.22-7.14 (m, 5H), 6.52 (s, I H), 6.46 (dt, J= 15.2, 7.3 Hz, IH), 6.28 (s, IH), 5.89 (t, J= 7.0 Hz, IH), 5.76 (s, IH), 5.59 (d, J= 15.2 Hz, IH), 5.37 (ddd, J= 15.2, 6.9, 6.9 Hz, I H), 5.24 (ddd, J= 15.2, 7.3, 7.0 Hz, I H), 2.56-2.49 (m, IH), 2.44-2.39 (m, IH), 2.01 - 1.92 (m, 4H); 13C NMR (100 MHz, C6D6, 25 0C): δ = 164.8, 162.8, 158.7, 153.1 , 137.6, 136.9, 136.1 , 133.9, 128.7 (x 2), 126.5 (x 2), 124.2, 122.0, 102.4, 100.8, 79.9, 39.0, 32.3, 31.4, four quaternary carbons are not detected; HRMS (ESI-TOF): m/z: calculated for C25H20ClF3O6Na: 531.0793, found 531.0992 [M+Na~].
Figure imgf000190_0001
2-153
Macrocycle 2-153: DHP (3.7 μL, 40.8 μmol) and PS-TsOH (12.7 mg, 40.8 μmol, 3.2 mmol.g" ') were added to a solution of compound 2-103 (12.9 mg, 40.8 μmol) in CH2Cl2 (1 mL) at 23 0C, and the mixture was stirred for 5 h. After this time, the reaction was filtered and the solvents were evaporated under reduced pressure. Purification by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) afforded 2-153 (13.8 mg, 85 %) as a mixture of two diastereoisomers. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 12.33 (s, IH), 12.1 1 (s, IH), 9.45 (s, I H), 9.40 (s. I H), 6.67, (m, 2H), 6.28 (2 x s, 2H), 5.83 (d, J= 13.2 Hz, IH), 5.79 (d, J = 12.9 Hz, I H), 5.35-5.30 (m, 3H), 5.27-5.22 (m, 3H), 5.06 (bd, J= 8.2 Hz, 2H), 4.10 (d, J = 17.5 Hz, 2H), 3.90-3.85 (m, IH), 3.80-3.76 (m, IH), 3.65 (d, J= 17.7 Hz, 2H), 3.57-3.52 (m, 2H), 3.46-3.41 (m, 2H), 2.77-2.71 (m, 3H), 2.53-2.49 (m, 3H), 2.36-2.29 (m, 4H), 2.24-1.56 (m, 12H), 1.31 (d, J = 6.4 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C23H28O6Na: 423.1778, found 423.1778 [M+Na~].
[00378] General procedure for the synthesis of compounds 2-154: The hydroxylamine R2ONH2 (5.0 equiv.) was added to a solution of compound 2-120 (1.0 equiv.) in pyridine/AcOH (5: 1 , 0.03 M) and the mixture was heated up to 40 0C. After stirring overnight, the solvents were evaporated under reduced pressure with silica gel. Purification over a short pad of silica gel with a mixture of 30 % EtOAc/cyclohexane afforded compound 2-154 (~99 %) as a mixture of two diastereoisomers cisl trans.
53412 v2 DC 189
Figure imgf000191_0001
Selected example of compounds 2-154: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.50-7.25 (m, 10H), 6.82 (s, I H), 6.75 (s, I H), 6.66 (s, I H), 6.48 (s, I H), 6.24-6.1 1 (m, 2H), 6.1 1-6.05 (m, 2H), 5.45-5.38 (m, 4H), 5.34-5.31 (m, 14 H), 4.50 (d, J = 17.2 Hz, IH), 3.65-3.38 (m, 8H), 3.60 (d, J = 17.1 Hz, I H), 3.54 (d, J - 17.1 Hz, IH), 3.24 (d, J = 17.2 Hz, I H), 2.48-2.36 (m, 4H), 2.21 -2.17 (m, 2H), 2.1 1-2.04 (m, 2H), 1.95-1.83 (m, 2H), 1.62-1.51 (m, 2H), 1.49 (d, J = 6.4 Hz, 6H), 1.32-1.20 (m, 12H); 13C NMR (100 MHz, CDCl3, 25 0C): δ = 168.1 , 167.9, 159.1 , 158.8, 157.2, 155.6, 155.4, 154.2, 140.8, 138.2, 138.2, 137.8, 136.9, 136.7, 132.3, 132.3, 128.3 (x 2), 128.3 (x 2), 128.2, 128.1 (x 2), 128.0 (x 2), 127.7, 127.6, 125.5, 1 18.8, 1 18.6, 1 18.3, 108.8, 108.5, 101.7, 101.7, 93.5, 93.4, 93.1 (x 2), 77.2, 76.0, 75.9, 71.2, 71.0, 64.5, 64.5, 64.3, 64.3, 40.0, 40.0, 34.9, 32.4, 32.3, 31.6, 31.1, 28.9, 20.3, 20.2, 15.0 (x 2), 15.0 (x 2); HRMS (ESI-TOF): m/z: calculated for C31H39NO7Na: 560.2619, found 560.2627 [M+Na+].
[00379] General procedure for the synthesis of compounds 2-155: PS-TsOH (10.0 equiv., 3.2 mmol.g"1) was added to a solution of compound 2-154 (1.0 equiv.) in MeOH (0.02 M) and the suspension was shaken at 40 0C for 4 h. After this time, the reaction mixture was filtered and the methanolic solution concentrated under reduced pressure. The crude product obtained was submitted without further purification to the next step. Thus, DHP (1.0 equiv.) and PS-TsOH (catalytic amount, 3.2 mmol.g"1) were added to a solution of this crude in CH2Cl2 (0.02 M ) at 23 0C, and the mixture was stirred for 5 h. After this time, the mixture was filtered, the solvents were evaporated under reduced pressure, and the remaining residue was purified by preparative TLC (silica gel, 30 % EtOAc/cyclohexane) to afford two different diastereoisomers 1 : 1 of 2-155 (~65 % yield).
Figure imgf000191_0002
[00380] Selected example of compounds 2-155: Less polar diastereoisomer: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 9.25 (s, IH), 9.24 (s, IH), 7.46-7.33 (m, 10H), 6.29 (s, IH), 6.26 (s,
53412 v2/DC 190 IH), 6.07-6.02 (m, 2H), 5.75 (d, J= 15.8 Hz, IH), 5.69 (d, J= 15.8 Hz, IH), 5.44-5.38 (m, 6H), 5.23 (s, 4H), 5.03 (d, J = 8.8 Hz, 2H), 4.34-4.13 (m, 6H), 3.69-3.63 (m, 2H), 2.70-2.67 (m, 2H), 2.30-2.16 (m, 6H), 2.08-1.94 (m, 8H), 1.73-1.65 (m, 8H), 1.42 (t, J= 6.4 Hz, 3H), 1.39 (t, J= 7.0 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C30H35NO6Na: 528.2357, found 528.2562 [Λ/+Na*].
[00381] More polar diastereoisomer: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.61 (s, IH), 9.27 (s, IH), 7.41 -7.33 (m, 5H), 6.62 (d, J= 16.4 Hz, IH), 6.47 (s, IH), 6.15-6.07 (m, IH), 5.50-5.38 (m, 3H), 5.16 (s, 2H), 5.04 (d, J = 10.5 Hz, IH), 4.30 (d, J= 15.2 Hz, IH), 4.24 (d, J = 10.5 Hz, IH), 3.84 (d, J= 15.2 Hz, IH), 3.66 (t, J= 11.4 Hz, IH), 2.71-2.65 (m, IH), 2.28-2.08 (m, 6H), 1.73-1.64 (m, 5H), 1.38 (t, J= 7.0 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C30H35NO6Na: 528.2357, found 528.2494 [M+Na+].
Figure imgf000192_0001
2-128
Macrocycle 2-128 from pochonin D (2-85): TBSCl (53.6 mg, 356 μmol) and imidazole (23.6 mg, 356 μmol) were added to a solution of pochonin D (2-85, 25 mg, 71.2 μmol) in DMF (5 mL) and the mixture was stirred for 3 h at room temperature. Purification by column chromatography (silica gel, 0-30 % EtOAc/cyclohexane gradient) afforded compound 2-128 (40 mg, 98 %).
[00382] General procedure for compounds 2-157: The hydroxylamine RONH2 (5.0 equiv.) was added to a solution of compound 2-128 (1.0 equiv.) in pyridine/AcOH (5: 1, 250 μL) and the mixture was heated up to 40 0C. After stirring overnight, the solvents were evaporated under reduced pressure, and filtration on silica gel with a mixture of 30 % EtOAc/cyclohexane afforded two isomers of 2-157 (~90 % yield).
Figure imgf000192_0002
[00383] Selected example of compounds 2-157: cis oxime: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.42 (bd, J = 6.4 Hz, 2H), 7.36 (bdd, J= 7.5, 6.9 Hz, 2H), 7.34-7.32 (m, IH), 6.52 (d, J= 16.1 Hz, IH), 6.38 (s, IH), 6.18-6.10 (m, IH), 5.36-5.32 (m, 2H), 5.16 (bs, 2H), 4.99-
53412 v2/DC 191 4.95 (m, I H), 3.79-3.76 (m, 2H), 2.40-1.99 (m, 6H), 1.45 (d, J= 6.2 Hz, 3H), 1.03 (s, 9H), 0.99 (s, 9H), 0.28 (s, 3H), 0.26 (s, 3H), 0.20 (s, 6H); trans oxime: 1H NMR (400 MHz, CDCh, 25 0C): δ = 7.44 (bd, J= 6.5 Hz. 2H), 7.37 (bdd, J = 7.6, 6.9 Hz, 2H), 7.33-7.31 (m, 1 H), 6.41 (s, 1 H), 6.04-5.97 (m, 1 H), 5.48 (bd, J = 15.0 Hz, 1 H), 5.29-5.27 (m, 1 H), 5.22 (bs, 2H), 5.00-4.95 (m, I H), 3.98-3.89 (m, 2H), 2.39-2.02 (m, 6H), 1.37 (d, J= 5.9 Hz, 3H), 1.04 (s, 9H), 0.99 (s, 9H), 0.28 (s, 3H), 0.27 (s, 3H), 0.23 (s, 3H), 0.22 (s, 3H). [00384] General procedure for compounds 2-158: To a solution of corresponding compound 2-157 (1.0 equiv) in THF was added TBAF (2.5 equiv, 1 M solution in THF) and the mixture was stirred at room temperature for 2 h. The solvents were then evaporated under reduced pressure, and filtration on silica gel with a mixture of 30 % EtOAc/cyclohexane afforded compound 2-158 m > 85 % yield.
Figure imgf000193_0001
[00385] Selected example of compounds 2-158: cis oxime: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1 .52 (s, 1 H), 7.45-7.34 (m, 5H), 6.64 (s, 1 H), 6.09-6.02 (m, 2H), 5.34-5.25 (m, 4H), 5.18-5.08 (m, 2H), 4.33 (d, J = 17.0 Hz, I H), 4.15 (d, J= 17.6 Hz, I H), 2.65-2.59 (m, IH), 2.27-2.14 (m, 3H), 2.04-2.00 (m, I H), 1.88-1.83 (m, IH), 1.30 (t, J = 6.4 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C25H26ClNO5Na: 478.1392, found 478.1372 [Λf+Naτ]. trans oxime: 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.73 (s, IH), 7.32-7,26 (m, 5H), 6.64 (s, I H), 6.50 (d, J = 16.4 Hz, I H), 6.06-5.98 (m, 2H), 5.43-5.24 (m, 3H), 4.91 (s, 2H), 4.22 (s, 2H), 2.61-2.55 (m, IH), 2.46-2.33 (m, 2H), 2.20-2.02 (m, 3H), 0.98 (t, J = 6.4 Hz, 3H); HRMS (ESI-TOF): m/z: calculated for C25H26ClNO5Na: 478.1392, found 478.1522 [M+Na+].
Figure imgf000193_0002
(±)-2-159
Compound (±)-2-159: A solution of cώ-butene oxide (1.75 mL, 20 mmol) in Et2O (10 mL) was cooled to -30 0C. Copper iodide (1.14 g, 6 mmol) was added to this solution and then, vinyl magnesium bromide (40 mL, I M solution in THF, 40 mmol) was added dropwise over a period of 1 h. The reaction mixture was then wanned up to room temperature over 12 h and the reaction turned black. The reaction mixture was quenched slowly with saturated NH4Claq
53412 v2 DC 192 (20 mL), stirred for 2 h, extracted with Et2O (20 mL) and dried over MgSO4. Concentration under reduced pressure afforded compound (±)-2-159 (1.3 g, 65 %). 1H NMR (400 MHz, CDCl3. 25 0C): δ = 5.81-5.72 (m, IH), 5.15 (d, J= 13.2 Hz, 2H), 3.59 (m, IH), 2.23-2.10 (m, I H), 1.21 (d, J= 6.4 Hz, 3H), 1.05 (d, J = 7.0 Hz, 3H).
Figure imgf000194_0001
Compound (±)-2-163: In a similar manner as that described for compound 2-112, compound (±)-2-163 was prepared with a 57 % yield from (±)-2-162. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 7.10 (s, I H), 6.76-6.71 (m, I H), 5.87 (d, J = 15.8 Hz, IH), 5.32 (s, 2H), 5.25-5.18 (s + m, 4H), 4.87-4.80 (m, I H), 3.99 (d, J = 16.9 Hz, IH), 3.80-3.71 (m, 5H), 2.32-2.26 (m, 2H), 2.20-2.1 1 (m, 3H), 1.36 (d, J= 6.4 Hz, 3H), 1.25 (t, J = 7.0 Hz, 6H), 1.04 (d, J= 7.0 Hz, 3H); 13C NMR (100 MHz. CDCl3, 25 0C): δ = 195.7, 166.6, 154.6, 153.8, 147.1 , 133.3, 132.8, 120.7, 1 17.7, 102.9, 93.9, 93.6, 76.2, 64.8, 64.6, 45.1 , 41.6, 30.7, 30.7, 18.2, 16.9, 15.0, 15.0.
Figure imgf000194_0002
(±)-2-164
Compound (±)-2-164: In a similar manner as that described for compound 2-85 using PS- TsOH, compound (±)-2-164 was prepared with a 40 % yield from (±)-2-163. 1H NMR (400 MHz, CDCl3, 25 0C): δ = 1 1.52 (s, IH), 6.78-6.69 (m, IH), 6.67 (s, IH), 6.05 (s, IH), 5.93 (d, J = 16.4 Hz, IH), 5.47-5.45 (m, IH), 5.37-5.33 (m, IH), 5.16 (dd, J = 15.8, 7.0 Hz, IH), 4.48 (bs, IH), 4.35 (d, J = 17.6 Hz, IH), 2.45-2.26 (m, 5H), 1.27 (d, J = 6.4 Hz, 3H), 1.10 (d, J = 7.0 Hz, 3H); ESI: m/∑: calculated for C18H2 IClO5: 365.12, found 365.22 [M+H+].
[00386] The description and examples provided herein are merely illustrative, and the invention is not so limited. Numerous variations, permutations and derivatives of these compounds, procedures and uses will occur to those of ordinary skill in the art, and are contemplated within the scope of the invention.
53412 v2 DC 193

Claims

We claim:
1. Use of a compound of formula I, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof, for the manufacture of a medicament for the treatment, prevention, or amelioration of tumors or symptoms resulting from neurofibromatosis in a subject suffering neurofibromatosis type 2 (NF2) or a condition associated with the loss of NF2 function or neurofibromatosis type 1 (NFl ) or a condition associated with the loss of NFl function,
Figure imgf000195_0001
wherein:
R1 . R2, R3, R4 and R5 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkylaryl, aralkyl, aryl, heteroalkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH2, NR2, SR, S(O)R, S(O)2R, -S(O)2NR2, -N(R)SO2R, -NC(O)R, - NC(O)NR2, -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR2, -OC(O)OR, or -
OC(O)NR2;
A1 and A2 together are -CH2-CH2-, -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, 1 ,2-cyclopropadiyl, or 1 ,2-oxirane;
B1 and B2 together are -CH2-CH2- or Bi and B2 together represent a covalent bond; X1 is hydrogen, halogen, OH, OR, NH2, NR2, NH-OR, SR, S(O)R, S(O)2R, -N-O-
(CH2)H-CO2-R; or X1 together with X2 or X3 represents a covalent bond;
X2 and X"' are both hydrogen, or one of X2 and X3 is hydrogen and the other together with X1 represents a covalent bond;
X4 and X5 together are =0, =S, =N-0R, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nCONR2, =N-NR2, =N-N-S(O)R or =N-N-S(O)2R; or one of X4 and X5 is hydrogen and the other is OH, OR, OC(O)R, OC(O)OR or OC(O)NR2; or one of X4 and X5 together with X6 represents a covalent bond and the other of X4 and X5 is OH, OR, OC(O)R, OC(O)OR, or OC(O)NR2;
53412 v2 DC 194 X6 is hydrogen or X6 together with one of X4 and X5 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl, heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is 0, 1 , 2 or 3.
2. Use of a compound according to claim 1, wherein the compound of formula I has the structure of formula II:
Figure imgf000196_0001
II wherein, R i 7' i ■s =0, =S, =N-OR, =N-O-(CH2)n-C(O)OR, =N-O-(CH2)n-C(O)NR2, =N-
NR2, -N-N-S(O)R or ^N-N-S(O)2R.
3. Use of a compound according to claim 1 or claim 2, wherein: R1 is H, Cl or heterocyclyl; R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are -CH-CH- or -C(OH)-C(OH)-; X1 together with X2 represent a bond; and
R7 is =0, =S, =N-OR, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N- S(O)R, =N-N-S(O)2R.
4. Use of a compound according to any of claims 1 to 3, wherein: R1 is H or Cl;
R3 is hydrogen;
R5 is hydrogen, methyl, propyl, isopropyl or phenyl; R7 is =N-0R, =N-O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2. ; and
53412 v2 DC 195 n -
5. Use of a compound according to any of claims 1 to 4, wherein R5 is hydrogen and R7 is =N-O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2.
6. Use of a compound according to any of claims 1 to 4, wherein R5 is hydrogen and R7 is =N-0R.
7. Use of a compound according to claim 1 or claim 2, wherein: R1 is H, Cl or heterocyclyl;
R" and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are 1,2-oxirane; X1 together with X2 represent a bond; and R7 is =0, =S, -N-OR, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, -N-NR2, =N-N-
S(O)R, =N-N-S(O)2R.
8. Use of a compound according to claim 1 or claim 2, wherein:
R1 is H, Cl or heterocyclyl; R2 and R4 are independently OH or OR;
R5 is hydrogen, alkyl, aryl or aralkyl;
A1 and A2 together are -CH=CH- or -C(OH)-C(OH)-;
X1 is hydrogen, halogen or NH-OR; and
R7 is =0, =S, =N-0R, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nC(O)NR2, -N-NR2, -N-N- S(O)R, =N-N-S(O)2R.
9. Use of a compound according to claim 1 or claim 2, wherein:
R1 is H, Cl or heterocyclyl; R2 and R4 are independently OH or OR; R3 is hydrogen, alkyl, aryl or aralkyl;
A1 and A2 together are 1,2-oxirane; X1 is hydrogen, halogen or NH-OR; and
R7 is =0, =S, =N-0R, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, -N-NR2, -N-N- S(O)R, -N-N-S(O)2R.
53412 v2/DC J 95
10, Use of a compound according to claim 1 , wherein the compound has the formula III:
Figure imgf000198_0001
wherein R is hydrogen, alkyl, arylalkyl, acyl or a protecting group.
1 1. Use of a compound according to claim 10, wherein X1 together with X2 represent a covalent bond.
12. Use of compound according to claim 10, wherein X1 is hydrogen, halogen, NH-OR NH- O-(CH2)nC(O)OR, or NH-O-(CHo)nC(O)NR2.
13. Use of a compound according to any of claims 10 to 12, wherein A1 and A2 together are -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, or 1 ,2- oxirane.
14. Use of a compound according to claim 1 , wherein the compound has the formula IV:
Figure imgf000198_0002
IV wherein R1 is hydrogen, OR, or NR2
53412 \2 DC 197
15. Use of a compound according to claim 14, wherein A1 and A2 together are -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, or 1 ,2-oxirane.
16. Use of a compound according to claim 1 , wherein the compound has the formula V:
Figure imgf000199_0001
V wherein R9 is (CH2)nC(O)OR, or -(CHa)nC(O)NR2; and n is O, 1 , 2 or 3.
17. Use of a compound according to claim 16, wherein R 9 i •s -CH2C(O)N(Me)OMe.
18. Use of a compound according to claim 16 or claim 17, wherein A1 and A2 together are -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, or 1,2- oxirane.
19. Use of a compound according to claim 1 , wherein the compound is selected from the group consisting of
Figure imgf000199_0002
53412 v2 'DC 198
Figure imgf000200_0001
Figure imgf000201_0001
53412 v2/DC 200
Figure imgf000202_0001
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000205_0002
53412 v2/'DC 204
Figure imgf000206_0001
Figure imgf000207_0001
53412 v2 DC 206
Figure imgf000208_0001
53412 v2/ DC 207
Figure imgf000209_0001
53412 v2/DC 208
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
53412 \2/DC 211
Figure imgf000213_0001
53412 v2/DC 212
Figure imgf000214_0001
Figure imgf000215_0001
53412 v2 DC 214
Figure imgf000216_0001
Figure imgf000217_0001
53412 v2'DC 216
Figure imgf000218_0001
53412 v2/DC 217
Figure imgf000219_0001
HO O
O"
HO'
Cl
Figure imgf000220_0001
Figure imgf000220_0002
53412 v2 DC 219
Figure imgf000221_0001
53412 v2/ DC 220
Figure imgf000222_0001
Figure imgf000223_0001
20. Use of a compound according to any of claims 1 to 19, wherein the at least one compound of formula I inhibits or slows growth of one or more NF2-deficient tumors or
53412 v2/DC 222 NFl -deficient tumors, reduces the number of said tumors or inhibits and/or reduces associated symptoms as compared to no treatment with the at least one compound of formula I.
21 , Use of a compound according to any of claims 1 to 20, wherein the at least one compound of formula I results in a decrease in size and/or number of said one or more NF2- deficient tumors.
22. Use of a compound according to any of claims 1 to 21 , wherein the one or more NF2- deficient tumors are selected from the group consisting of vestibular schwannomas; spinal cord schwannomas; sporadic schwannomas; peripheral nerve schwannomas; schwannoma; meningioma; mesothelioma; ependymoma; glioma and astrocytoma.
23. Use of a compound according to any of claims 1 to 22, wherein the vestibular schwannomas comprises a unilateral vestibular schwannoma or a bilateral vestibular schwannoma.
24. Use of a compound according to any of claims 1 to 23, wherein the at least one compound of formula I decreases size and/or number of said one or more NFl -deficient tumors.
25. Use of a compound according to any of claims 1 to 24, wherein said one or more NFl - deficient tumors are selected from the group consisting of dermal and plexiform neurofibromas, optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ependymomas, pheochromocytomas and ganglioneuromas, rhabdomyosarcomas, neurofibrosarcomas, malignant peripheral nerve sheath tumors ("MPNST"), malignant schwannomas, and JMML.
26. Use of a compound according to any of claims 1 to 25, wherein the at least one compound of formula I reduces the function of HSP90 complex and/or the upregulates or increases in HSP70 in said subject.
53412 v2 DC 223
27. Use of a compound according to any of claims 1 to 26, wherein the at least one compound of formula I improves at least one of the subject's hearing, balance and vision; or increases in muscle mass; or reduces tumor burden in the subject.
28. Use of a compound according to any of claims 1 to 27, wherein the reduction in tumor burden is identified using a MRI or a CAT scan.
29. Use of a compound according to any of claims 1 to 28, wherein the compound of formula I is administered orally, intravenously or locally, such as intralesionally or topically.
30. Use of a compound according to any of claims 1 to 29, wherein the compound of formula I is administered before, during or after surgical removal of a tumor.
31. Use of a compound according to any of claims 1 to 30, further comprises at least one additional active agent.
32. Use of a compound according to any of claims 1 to 31, wherein the at least one additional active agent comprises a heat shock protein 90 (HSP90) inhibitor or a cytotoxic agent.
33. Use of a compound according to claim 32, wherein the HSP90 inhibitor is selected from the group consisting of (1) 17-AAG or a derivative thereof; (2) a purine scaffold-based HSP90 inhibitor or a derivative thereof; (3) a pyrazole scaffold-based HSP90 inhibitor or a derivative thereof; (4) an imidazole scaffold-based HSP90 inhibitor or a derivative thereof; (5) a tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitor or a derivative thereof; (6) Novobiocin or a derivative thereof; (7) NZ28 (NCS- 134754) or a derivative thereof ; (8) a HSP90 modulator; (9) a biological agent or molecule; and (10) a combination thereof.
34. Use of a compound according to claim 33, wherein the biological agent or molecule is selected from the group consisting of peptide, antibody, siRNA, antisense nucleic acid molecule, and a combination thereof.
35. Use of a compound according to claim 33, wherein the HSP90 modulator is selected from the group consisting of a HDAC inhibitor, Trichostatin A or a derivative thereof, SAHA
53412 v2 DC 224 or a derivative thereof, LAQ824 or a derivative thereof, FK228 or a derivative thereof, and a combination thereof.
36. A method of inhibiting or reducing the growth or number of NF2-deficient tumor cells or NFl -deficient tumor cells comprising contacting said NF2-deficient tumor cells or NFl - deficient tumor cells with at least one compound of formula I, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof:
Figure imgf000226_0001
I wherein:
R1, R2, R3, R4 and R5 are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkylaryl, aralkyl, aryl, heteroalkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH2, NR2, SR, S(O)R, S(O)2R, -S(O)2NR2, - N(R)SO2R, -NC(O)R, -NC(O)NR2, -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, - C(O)NR2, -OC(O)OR, or -OC(O)NR2;
A1 and A2 together are -CH2-CH2-, -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, 1 ,2-cyclopropadiyl, or 1,2-oxirane;
B and B" together are -CH2-CH2- or Bi and B2 together represent a covalent bond;
X1 is hydrogen, halogen, OH, OR, NH2, NR2, NH-OR, SR, S(O)R, S(O)2R, -N-O-(CH2)n-CO2-R; or X1 together with X2 or X3 represents a covalent bond;
X and X3 are both hydrogen, or one of X2 and X3 is hydrogen and the other together with X1 represents a covalent bond;
X4 and X5 together are =0, =S, =N-0R, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nCONR2, =N-NR2, =N-N-S(0)R or -N-N-S(O)2R; or one of X4 and X5 is hydrogen and the other is OH, OR, OC(O)R, OC(O)OR or OC(O)NR2; or one of X4
53412 v2'DC 225 and X5 together with X6 represents a covalent bond and the other of X4 and X5 is OH, OR, OC(O)R, OC(O)OR, or OC(O)NR2;
X6 is hydrogen or X6 together with one of X4 and X5 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl, heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is O, 1, 2 or 3.
37. The method of claim 36, wherein the compound of formula I has the structure of formula II:
Figure imgf000227_0001
II wherein, R > 7' i •s =0, =S, =N-0R, =N-O-(CH2)n-C(O)OR, =N-O-(CH2)n-C(O)NR2, =N- NR2, =N-N-S(O)R or =N-N-S(O)2R.
38. The method of claim 36 or claim 37, wherein: R1 is H, Cl or heterocyclyl;
R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are -CH=CH- or -C(OH)-C(OH)-; X1 together with X2 represent a bond; and R7 is =0, =S, =N-0R, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nC(O)NR2, ^N-NR2, -N-N-
S(O)R, =N-N-S(O)2R.
39. The method of any of claims 36 to 38, wherein:
53412 v2/ DC 226 R1 is H or Cl;
R3 is hydrogen;
R5 is hydrogen, methyl, propyl, isopropyl or phenyl;
R7 is =N-OR, =N-O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2. ; and n = 1.
40. The method of any of claims 36 to 39, wherein R'"1 is hydrogen and R7 is =N- O-(CH2)nC(O)OR, or =N-O-(CH2)nC(O)NR2.
41. The method of any of claims 36 to 39, wherein R5 is hydrogen and R7 is =N-OR.
42. The method of any of claims 36 to 41 , wherein:
R1 is H, Cl or heterocyclyl;
R2 and R4 are independently OH or OR; R' is hydrogen, alkyl, aryl or aralkyl;
A1 and A2 together are 1 ,2-oxirane;
X1 together with X2 represent a bond; and
R7 is =0, =S, -N-OR, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N-
S(O)R, =N-N-S(0)2R.
43. The method of any of claims 36 to 42, wherein:
R1 is H, Cl or heterocyclyl; R2 and R4 are independently OH or OR; R'"1 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are -CH=CH- or -C(OH)-C(OH)-;
X1 is hydrogen, halogen or NH-OR; and
R7 is =0, =S, =N-0R, =N-0-(CH2)nC(0)0R, =N-O-(CH2)nC(O)NR2, =N-NR2, -N-N- S(O)R, -N-N-S(O)2R.
44. The method of any of claims 36 to 43, wherein: R1 is H, Cl or heterocyclyl; R2 and R4 are independently OH or OR; R5 is hydrogen, alkyl, aryl or aralkyl; A1 and A2 together are 1,2-oxirane;
53412 v2'DC 227 X1 is hydrogen, halogen or NH-OR; and
R7 is =0, =S, =N-OR, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nC(O)NR2, =N-NR2, =N-N- S(O)R, =N-N-S(O)2R.
45, The method of claim 36, wherein the compound has the formula III:
Figure imgf000229_0001
wherein R8 is hydrogen, alkyl, arylalkyl, acyl or a protecting group.
46. The method of claim 45, wherein X1 together with X2 represent a covalent bond.
47. The method of claim 45 or claim 46, wherein X1 is hydrogen, halogen, NH-OR NH- O-(CH2)nC(O)OR, or NH-O-(CH2)nC(O)NR2.
48. The method of any of claims 36 to 47, wherein A1 and A2 together are -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, or 1 ,2-oxirane.
49. The method of claim 36, wherein the compound has the formula IV:
Figure imgf000229_0002
IV wherein R' is hydrogen, OR, or NR2.
53412 v2/ DC 228
50. The method of claim 49, wherein A1 and A2 together are -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, or 1 ,2-oxirane.
51 , The method of claim 36, wherein the compound has the formula V:
Figure imgf000230_0001
V wherein R .9' i •s (CHo)nC(O)OR, or -(CHa)nC(O)NR2; and n is 0, 1 , 2 or 3.
52. The method of claim 51 , wherein R9 is -CH2C(O)N(Me)OMe.
53. The method of claim 51 or claim 52, wherein A1 and A2 together are -CH=CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, or 1 ,2-oxirane.
54. The method of claim 36, wherein the compound is selected from the group consisting of
Figure imgf000230_0002
53412 v2/DC 229
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000232_0002
53412 v2/ DC 231
Figure imgf000233_0001
53412 v2/DC 232
Figure imgf000234_0001
Figure imgf000234_0002
53412 v2/DC 233
Figure imgf000235_0001
534 ] 2 v2 DC 234
Figure imgf000236_0001
53412 v2/DC 235
Figure imgf000237_0001
Figure imgf000237_0002
Figure imgf000237_0003
53412 v2 DC 236
Figure imgf000238_0001
Figure imgf000239_0001
53412 v2'DC 238
Figure imgf000240_0001
Figure imgf000241_0001
53412 v2'DC 240
Figure imgf000242_0001
Figure imgf000243_0001
53412 v2'DC 242
Figure imgf000244_0001
Figure imgf000245_0001
53412 v2'DC 244
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
53412 v2 DC 247
Figure imgf000249_0001
53412 v2 'DC 248
Figure imgf000250_0001
EOMO O
O"
Figure imgf000250_0002
EOMO O
O
Figure imgf000250_0003
53412 v2/ DC 249 HO O
O
Figure imgf000251_0001
53412 v2'DC 250
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
53412 v2'DC 253
55. The method of any of claims 36 to 54, wherein the contact of said NF2-deficient tumor cells or NFl -deficient tumor cells with said at least one compound of formula I occurs in vitro, in vivo, or ex vivo.
56. The method of any of claims 36 to 55, wherein said NF2-deficient tumor cells are Nf2- deficient mouse Schwann cells or NF2-defϊcient human schwannoma cells; and said NFl- deficient tumor cells are NfI -deficient mouse Schwann cells or NFl -deficient human Schwann cells.
57. The method of any of claims 36 to 56, wherein said NF2-deficient tumor cells or said NFl -deficient tumor cells are from a human, canine, rat or mouse.
58. The method of any of claims 36 to 57, wherein said NF2-deficient tumor cells are selected from the group consisting of NF2-deficient schwannoma cell line cells, NF2- deficient meningioma cell line cells and NF2-deficient mesothelioma cell line cells.
59. The method of any of claims 36 to 58, wherein said NF2-deficient tumor cells are selected from the group consisting of HEI 193 cells, SFl 335 cells, BAR cells and RAV cells.
60. The method of any of claims 36 to 54, wherein said NFl -deficient tumor cells are selected from the group consisting of human MPNST cells, primary neurofibroma cells derived from NFl patients, mouse /V/7,;p53-deficient MPNST cell lines established from cisNfl;p53 mice, and NfI-/- mouse cells, such as Schwann cells, mouse embryonic cells, and leukemia cells.
61. The method of any of claims 36 to 54 and 60, wherein said NFl-deficient tumor cells are selected from the group consisting of ST88-14, 88-3, 90-8, and sNF96.2.
62. The method of any of claims 36 to 61, wherein contact of said NF2-deficient tumor cells or said NFl -deficient tumor cells with said at least one compound of formula I results in a reduction in HSP90 function and/or an upregulation of HSP70.
63. The method of any of claims 36 to 62, wherein contact of said NF2-deficient tumor cells or said NFl -deficient tumor cells with said at least one compound of formula I results in
53412 v2< DC 254 degradation of ErbB2 and/or phosphorylated ErbB2; degradation of Akt and/or phosphorylated Akt; degradation of Raf and/or phosphorylated Raf; or a reduction in phosphorylation of proteins downstream of the ErbB2, Akt or Raf signaling pathway.
64, The method of claim 63, wherein said degradation or upregulation of the proteins or reduction in phosphorylated proteins is detected using an antibody.
65. The method of any of claims 36 to 66, further comprises contacting said NF2-deficient tumor cells or NFl -deficient tumor cells with at least one additional active agent.
66. The method of claim 65, wherein the contact of said NF2-deficient tumor cells or NFl- deficient tumor cells with at least one additional active agent and the contact of said NF2- deficient tumor cells or NFl -deficient tumor cells with at least one compound of formula I occur simultaneously or sequentially.
67. The method of any of claims 36 to 66, wherein the at least one additional active agent comprises a heat shock protein 90 (HSP90) inhibitor or a cytotoxic agent.
68. The method of claim 67, wherein the HSP90 inhibitor is selected from the group consisting of (1 ) 17-AAG or a derivative thereof; (2) a purine scaffold-based HSP90 inhibitor or a derivative thereof; (3) a pyrazole scaffold-based HSP90 inhibitor or a derivative thereof; (4) an imidazole scaffold-based HSP90 inhibitor or a derivative thereof; (5) a tetrahydroindolone- or tetrahydroindazolone-based HSP90 inhibitor or a derivative thereof; (6) Novobiocin or a derivative thereof; (7) NZ28 (NCS- 134754) or a derivative thereof ; (8) a HSP90 modulator; (9) a biological agent or molecule; and (10) a combination thereof.
69. The method of claim 68, wherein the biological agent or molecule is selected from the group consisting of peptide, antibody, siRNA, antisense nucleic acid molecule, and a combination thereof.
70. The method of claim 68, wherein the HSP90 modulator is selected from the group consisting of a HDAC inhibitor, Trichostatin A or a derivative thereof, SAHA or a derivative thereof, LAQ824 or a derivative thereof, FK228 or a derivative thereof, and a combination thereof.
53412 v2/DC 255 71 , Use of a compound of formula I, or a pharmaceutically acceptable tautomer, salt, solvate, ester, and/or prodrug thereof, for the manufacture of a medicament for the treatment, prevention, or amelioration of a neurodegenerative disease,
Figure imgf000257_0001
I wherein:
R1, R2, R3, R4 and RΛ are independently hydrogen, halogen, nitro, cyano, alkyl, lower alkyl, alkenyl, alkynyl, alkyl aryl, aralkyl, aryl, heteroalkyl, alkylheteroaryl, heterocyclyl, heteroaryl, OH, OR, NH2, NR2, SR, S(O)R, S(O)2R, -S(O)2NR2, -N(R)SO2R, -NC(O)R, -
NC(O)NR2, -NC(O)OR, -OC(O)R, -C(O)R, -C(O)OR, -C(O)NR2, -OC(O)OR, or -
OC(O)NR2;
A1 and A2 together are -CH2-CH2-, -CH-CH-, -CH(OH)-CH(OH)-, -CH(OH)-CH(halogen)-, -CH(halogen)-CH(OH)-, 1 ,2-cyclopropadiyl, or 1,2-oxirane; B1 and B2 together are -CH2-CH2- or Bi and B2 together represent a covalent bond;
X1 is hydrogen, halogen, OH, OR, NH2, NR2, NH-OR, SR, S(O)R, S(O)2R, -N-O- (CH2)H-CO2-R; or X1 together with X2 or X3 represents a covalent bond;
X2 and X3 are both hydrogen, or one of X2 and X3 is hydrogen and the other together with X1 represents a covalent bond; X4 and X5 together are =0, =S, =N-0R, =N-O-(CH2)nC(O)OR, =N-O-(CH2)nCONR2,
=N-NR2, =N-N-S(0)R or =N-N-S(0)2R; or one of X4 and X5 is hydrogen and the other is OH, OR, OC(O)R, OC(O)OR or OC(O)NR2; or one of X4 and X5 together with X6 represents a covalent bond and the other of X4 and X5 is OH, OR, OC(O)R, OC(O)OR, or OC(O)NR2;
X6 is hydrogen or X6 together with one of X4 and X5 represents a covalent bond; each R is independently the same or different, and is hydrogen, alkyl, lower alkyl, acyl including acetyl and trifluoroacetyl, aryl, alkaryl, arylalkyl including benzyl, heteroalkyl,
53412 v2/DC 256 heteroaryl, heterocyclyl, or a protecting group; or two R on the same nitrogen are taken together with the nitrogen to form a 5-8 membered heterocyclic or heteroaryl ring; and n is 0, 1 , 2 or 3.
53412 v2'DC 257
PCT/US2007/075739 2006-06-02 2007-08-10 Treatment of neurofibromatosis with radicicol and its derivatives WO2008150302A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA2727100A CA2727100C (en) 2006-06-02 2007-08-10 Treatment of neurofibromatosis with radicicol and its derivatives
US12/631,667 US8329683B2 (en) 2006-06-02 2009-12-04 Treatment of neurofibromatosis with radicicol and its derivatives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US2007/070367 WO2007143630A2 (en) 2006-06-02 2007-06-04 Treatment of neurofibromatosis with hsp90 inhibitors
USPCT/US2007/070367 2007-06-04

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/070367 Continuation-In-Part WO2007143630A2 (en) 2006-06-02 2007-06-04 Treatment of neurofibromatosis with hsp90 inhibitors

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/631,667 Continuation-In-Part US8329683B2 (en) 2006-06-02 2009-12-04 Treatment of neurofibromatosis with radicicol and its derivatives

Publications (1)

Publication Number Publication Date
WO2008150302A1 true WO2008150302A1 (en) 2008-12-11

Family

ID=38984220

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/075739 WO2008150302A1 (en) 2006-06-02 2007-08-10 Treatment of neurofibromatosis with radicicol and its derivatives

Country Status (1)

Country Link
WO (1) WO2008150302A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011004132A1 (en) 2009-07-10 2011-01-13 Sanofi-Aventis Novel hsp90-inhibiting indole derivatives, compositions containing said derivatives, and use thereof
WO2011027081A2 (en) 2009-09-03 2011-03-10 Sanofi-Aventis Novel derivatives of 5,6,7,8-tetrahydroindolizine inhibiting hsp90, compositions containing same, and use thereof
WO2010141738A3 (en) * 2009-06-03 2011-03-17 President And Fellows Of Harvard College Compositions and method for inhibiting tumor growth
JP2016533393A (en) * 2013-09-30 2016-10-27 パテオン エーピーアイ サービシーズ インコーポレイテッドＰａｔｈｅｏｎ Ａｐｉ Ｓｅｒｖｉｃｅｓ Ｉｎｃ． A new synthetic route to prostaglandins and prostaglandin intermediates using metathesis
US10239856B2 (en) * 2008-01-15 2019-03-26 University De Strasbourg Synthesis of resorcylic acid lactones useful as therapeutic agents

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001003692A1 (en) * 1999-07-09 2001-01-18 Oregon Health Sciences University Compositions and methods for promoting nerve regeneration
WO2002016369A2 (en) * 2000-08-25 2002-02-28 Sloan-Kettering Institute For Cancer Research Novel macrocycles and uses thereof
WO2006083979A2 (en) * 2005-02-02 2006-08-10 Nexgenix Pharmaceuticals, L.L.C. Local treatment of neurofibromas
WO2007143630A2 (en) * 2006-06-02 2007-12-13 Nexgenix Pharmaceuticals Treatment of neurofibromatosis with hsp90 inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001003692A1 (en) * 1999-07-09 2001-01-18 Oregon Health Sciences University Compositions and methods for promoting nerve regeneration
WO2002016369A2 (en) * 2000-08-25 2002-02-28 Sloan-Kettering Institute For Cancer Research Novel macrocycles and uses thereof
WO2006083979A2 (en) * 2005-02-02 2006-08-10 Nexgenix Pharmaceuticals, L.L.C. Local treatment of neurofibromas
WO2007143630A2 (en) * 2006-06-02 2007-12-13 Nexgenix Pharmaceuticals Treatment of neurofibromatosis with hsp90 inhibitors

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHAUDHURY SUBHABRATA ET AL.: "Hsp90 as a target for drug developement", CHEMMEDCHEM, vol. 1, no. 12, December 2006 (2006-12-01), pages 1331 - 1340, XP002467848 *
IKUINA Y ET AL: "Synthesis and antitumor activity of novel O-carbamoylmethyloxime derivatives of radicicol", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 46, no. 12, 2003, pages 2534 - 2541, XP002975841, ISSN: 0022-2623 *
SANO M ET AL: "RADICICOL POTENTIATES NEUROTROPHIN-MEDIATED NEURITE OUTGROWTH AND SURVIVAL OF CULTURED SENSORY NEURONS FROM CHICK EMBRYO", JOURNAL OF NEUROCHEMISTRY, NEW YORK, NY, US, vol. 72, June 1999 (1999-06-01), pages 2256 - 2263, XP002931836, ISSN: 0022-3042 *
SHIOTSU YUKIMASA ET AL: "Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G1 phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex", BLOOD, W.B.SAUNDERS COMPANY, ORLANDO, FL, US, vol. 96, no. 6, 15 September 2000 (2000-09-15), pages 2284 - 2291, XP002190760, ISSN: 0006-4971 *
SOGA S ET AL: "KF25706, a Novel Oxime Derivative of Radicicol, Exhibits in Vivo Antitumor Activity via Selective Depletion of Hsp90 Binding Signaling Molecules", DIALOG (CANCERLIT), 15 June 1999 (1999-06-15), XP002997357 *
SOGA S ET AL: "STEREOSPECIFIC ANTITUMOR ACTIVITY OF RADICICOL OXIME DERIVATIVES", CANCER CHEMOTHERAPY AND PHARMACOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 48, no. 6, December 2001 (2001-12-01), pages 435 - 445, XP001153416, ISSN: 0344-5704 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10239856B2 (en) * 2008-01-15 2019-03-26 University De Strasbourg Synthesis of resorcylic acid lactones useful as therapeutic agents
WO2010141738A3 (en) * 2009-06-03 2011-03-17 President And Fellows Of Harvard College Compositions and method for inhibiting tumor growth
WO2011004132A1 (en) 2009-07-10 2011-01-13 Sanofi-Aventis Novel hsp90-inhibiting indole derivatives, compositions containing said derivatives, and use thereof
WO2011027081A2 (en) 2009-09-03 2011-03-10 Sanofi-Aventis Novel derivatives of 5,6,7,8-tetrahydroindolizine inhibiting hsp90, compositions containing same, and use thereof
JP2016533393A (en) * 2013-09-30 2016-10-27 パテオン エーピーアイ サービシーズ インコーポレイテッドＰａｔｈｅｏｎ Ａｐｉ Ｓｅｒｖｉｃｅｓ Ｉｎｃ． A new synthetic route to prostaglandins and prostaglandin intermediates using metathesis

Similar Documents

Publication Publication Date Title
EP2136799B1 (en) Macrocyclic compounds useful as inhibitors of kinases and hsp90
CA2727100C (en) Treatment of neurofibromatosis with radicicol and its derivatives
CN110114071B (en) Tricyclic compounds containing substituted pyridones and methods of using the same
JP2022174114A (en) Alk protein decomposer and use thereof in cancer therapy
AU2011319685B2 (en) Pochoxime conjugates useful for the treatment of HSP90 related pathologies
EA023493B1 (en) Pyrazoloquinoline compound
JPWO2002060890A1 (en) New physiologically active substance
WO2008150302A1 (en) Treatment of neurofibromatosis with radicicol and its derivatives
US8329683B2 (en) Treatment of neurofibromatosis with radicicol and its derivatives
EP2173733A2 (en) Compositions and methods comprising analogues of radicicol a
KR101640951B1 (en) Macrocyclic prodrug compounds useful as therapeutics
JPWO2008133288A1 (en) Tricyclic aryl compounds
US11834463B2 (en) High affinity macrocyclic FKB51-inhibitors for treatment of psychiatric disorders
CN112218878A (en) NTCP inhibitors
US20230287031A1 (en) New glycolipids and use thereof as sk3 ion channel modulators
US20230139593A1 (en) Treatment of disorders associated with oxidative stress and compounds for same
WO2023248010A2 (en) Targeted modulators of jak3 for treatment of inflammatory and autoimmune diseases
WO2005061481A1 (en) Novel ma crocycles and uses thereof
US20110190237A1 (en) Macrocyclic Prodrug Compounds Useful as Therapeutics

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07800083

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2010511153

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: JP

122 Ep: pct application non-entry in european phase

Ref document number: 07800083

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2727100

Country of ref document: CA